Treatment regimens using anti-nkg2a antibodies

ABSTRACT

The present invention relates to methods for the treatment of disease, notably cancer, using antibodies that specifically bind and inhibit human NKG2A. Included are therapeutic regimens that provide improved efficacy of anti-NKG2A antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/511,792, filedMar. 16, 2017, now U.S. Pat. No. 10,676,523, which is the U.S. nationalstage application of International Patent Application No.PCT/EP2015/071073, filed Sep. 15, 2015, which claims the benefit of U.S.Provisional Application Nos. 62/050,948, filed Sep. 16, 2014; 62/067,642filed Oct. 23, 2014; 62/083,929 filed Nov. 25, 2014; 62/093,141 filedDec. 17, 2014; and 62/093,124 filed Dec. 17, 2014; all of which areincorporated herein by reference in their entirety; including anydrawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“NKG2A-T_ST25”, created Sep. 15, 2015, which is 26 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the use of anti-NKG2A-antibodies for therapy,notably for the treatment of cancers.

BACKGROUND OF THE INVENTION

NK cell activity is regulated by a complex mechanism that involves bothactivating and inhibitory signals. Several distinct NK-specificreceptors have been identified that play an important role in the NKcell mediated recognition and killing of HLA Class I deficient targetcells. Natural Cytotoxicity Receptors (NCR) refers to a class ofactivating receptor proteins, and the genes expressing them, that arespecifically expressed in NK cells. Examples of NCRs include NKp30,NKp44, and NKp46 (see, e.g., Lanier (2001) Nat Immunol 2:23-27, Pende etal. (1999) J Exp Med. 190:1505-1516, Cantoni et al. (1999) J Exp Med.189:787-796, Sivori et al (1997) J. Exp. Med. 186:1129-1136, Pessino etal. (1998) J Exp Med. 188(5):953-60; Mandelboim et al. (2001) Nature409:1055-1060, the entire disclosures of which are herein incorporatedby reference). These receptors are members of the Ig super-family, andtheir cross-linking, induced by specific mAbs, leads to a strong NK cellactivation resulting in increased intracellular Ca⁺⁺ levels, triggeringof cytotoxicity, and lymphokine release, and an activation of NKcytotoxicity against many types of target cells. CD94/NKG2A is aninhibitory receptor found on subsets of natural killer cells (NK cells),Natural Killer T cells (NKT cells) and T cells (α/β and γ/δ). CD94/NKG2Arestricts cytokine release and cytotoxic responses of aforementionedlymphocytes towards cells expressing the CD94/NKG2A-ligand HLA-E (see,e.g., WO99/28748). HLA-E has also been found to be secreted in solubleform by certain tumor cells (Derre et al., J Immunol 2006; 177:3100-7)and activated endothelial cells (Coupel et al., Blood 2007;109:2806-14). Antibodies that inhibit CD94/NKG2A signalling may increasethe cytokine release and cytolytic activity of lymphocytes towards HLA-Epositive target cells, such as responses of CD94/NKG2A-positive NK cellsresponses towards virally infected cells. Therefore, therapeuticantibodies that inhibit CD94/NKG2A but that do not provoke the killingof CD94/NKG2A-expressing cells (i.e. non-depleting antibodies), mayinduce control of tumor-growth in cancer patients. In addition,anti-NKG2A antibodies have also been suggested for use in treatingautoimmune or inflammatory diseases (see, e.g., US20030095965,WO2006070286).

Various antibodies against NKG2A have been described in the art.WO2006070286 and U.S. Pat. No. 8,206,709 (see also WO2008/009545)describe anti-NKG2A antibody Z270, while WO2009/092805 describeshumanized anti-NKG2A antibody Z199. Vance et al. (J Exp Med 1999; 190:1801-12) refers to rat anti-murine NKG2-antibody 20D5 (now commerciallyavailable via BD Biosciences Pharmingen, Catalog No. 550518, USA); andU.S. patent application publication 20030095965 describes murineantibody 3S9. Antibody Z270 binds and neutralizes the inhibitoryreceptor NKG2A without neutralizing the activating receptors NKG2C andNKG2E. Antibody Z199, 20D5 and 3S9 all bind the activating NKG2 familymembers NKG2C and NKG2E in addition to NKG2A. Antibody Z270 blocks thebinding of HLA-E to NKG2A, while antibody Z199 neutralises NKG2A withoutinterfering with the binding of NKG2A to HLA-E.

SUMMARY OF THE INVENTION

The present inventors have discovered that doses and concentrations ofanti-NKG2A antibody that fully occupy NKG2A receptors on lymphocytes donot yield maximal neutralization of NKG2A receptors in the presence ofHLA-E expressing target cells in vivo. In particular, the concentrationof anti-NKG2A antibody required for full neutralization of NKG2Areceptors on NK cells in the presence of HLA-E expressing target cellsis 100-fold higher than the concentration observed to fully occupy NKG2Areceptors in binding assays using NKG2A+ lymphocytes. In parallel, it isobserved that the anti-tumor effect of anti-NKG2A is increased as afunction of higher expression of HLA-E on tumor cells.

As shown herein NKG2A+ NK and CD8+ T lymphocytes are present not only incirculation but within the tumor environment, and NKG2A+ CD8 T cells mayfurthermore be found at significantly increased frequencies within thetumor-infiltrating subset, compared to CD8+ T cells in tumor draininglymph nodes and spleen. The treatment regimens of the inventiontherefore additionally provide advantageous methods for treating solidtumors by using anti-NKG2A antibodies to modulate lymphocytes within thetumor environment.

In one embodiment, provided is a method for treating or preventing adisease (e.g. a cancer, a solid tumor, a hematological tumor) in anindividual, the method comprising administering to an individual havingdisease (e.g. a cancer, a solid tumor) an antibody that neutralizes theinhibitory activity of a human NKG2A polypeptide, wherein the anti-NKG2Aantibody is administered in an amount effective to achieve a blood(serum) concentration of anti-NKG2A antibody that corresponds to atleast the EC₅₀, optionally the EC₁₀₀, for NKG2A+ NK cell response. Inone embodiment, the amount is effective to achieve a concentration inextravascular tissue (e.g. tumor tissue) of anti-NKG2A antibody thatcorresponds to at least the EC₅₀, optionally the EC₁₀₀, for NKG2A+ NKcell response. In one embodiment, NKG2A+ NK cell response is assessedusing an assay of cytotoxic activity of NKG2A-expressing NK cells towardHLA-E expressing target cells.

In one embodiment, provided is a method for treating or preventing adisease (e.g. a cancer, a solid tumor, a hematological tumor) in anindividual, the method comprising administering to an individual havingdisease (e.g. a cancer, a solid tumor) an antibody that neutralizes theinhibitory activity of a human NKG2A polypeptide, wherein the anti-NKG2Aantibody is administered in an amount effective to achieve (and/or tomaintain for a specified period of time or between two successiveadministrations) a blood (serum) concentration of anti-NKG2A antibody ofat least 10 μg/ml (or, optionally at least 20, 30, 40, 50, 80 or 100μg/m L).

Accordingly, in one embodiment, provided is a method for treating orpreventing a disease (e.g. a cancer, a solid tumor, a hematologicaltumor) in an individual, the method comprising administering to anindividual an antibody that binds NKG2A and that neutralizes theinhibitory activity of a human NKG2A polypeptide for at least oneadministration cycle, the administration cycle comprising at least afirst and second (and optionally a 3^(rd), 4^(th), 5^(th), 6^(th),7^(th), and/or 8^(th) or further) administration of the anti-NKG2Aantibody, wherein the anti-NKG2A antibody is administered in an amounteffective to achieve blood concentration of anti-NKG2A antibody, and/orto maintain blood concentration of anti-NKG2A antibody between twosuccessive (e.g. said first and second, and optionally the further)administrations, which concentration is at least 10-fold (e.g., 10-20fold, 10-50 fold, 10-100 fold, 20-50 fold, 20-100 fold, 30-100 fold,50-100 fold), optionally at least 50-, 60-, 80- or 100-fold, the minimumconcentration required to substantially fully (e.g. 90%, 95%) occupy(saturate) NKG2A receptors on the surface of NKG2A+ cells (e.g., asassessed by titrating anti-NKG2A antibody on NKG2A-expressing cells inPBMC). In one embodiment, the anti-NKG2A antibody competes with HLA-Efor binding to human NKG2A.

In one embodiment, provided is a method for treating or preventing adisease (e.g. a cancer, a solid tumor, a hematological tumor) in anindividual, the method comprising administering to an individual havingdisease (e.g. a cancer, a solid tumor) an antibody that neutralizes theinhibitory activity of a human NKG2A polypeptide for at least oneadministration cycle, the administration cycle comprising at least afirst and second (and optionally a 3^(rd), 4^(th), 5^(th), 6^(th),7^(th) and/or 8^(th) or further) administration of the anti-NKG2Aantibody, wherein the anti-NKG2A antibody is administered in an amounteffective to achieve, or to maintain between two successiveadministrations, a blood (serum) concentration of anti-NKG2A antibody ofat least 10 μg/ml (or, optionally at least 20, 30, 40 or 50 μg/mL). Inone embodiment, a specified continuous blood concentration ismaintained, wherein the blood concentration does not drop substantiallybelow the specified blood concentration for the duration of thespecified time period (e.g. between two administrations of antibody,number of weeks), i.e. although the blood concentration can vary duringthe specified time period, the specified blood concentration maintainedrepresents a minimum or “trough” concentration. In one embodiment, atherapeutically active amount of an anti-NKG2A antibody is an amount ofsuch antibody capable of providing (at least) the EC₅₀ concentration,optionally substantially the EC₁₀₀ concentration, in blood and/or in atissue for NKG2A+ NK cell response for a period of at least about 1week, about 2 weeks, or about one month, following administration of theantibody. In one embodiment, the anti-NKG2A antibody is administered inan amount effective to achieve blood (serum) concentration of anti-NKG2Aantibody of at least 10 μg/ml (or, optionally at least 20, 30, 40 or 50μg/mL) for a period of at least about 1 week, at least about 2 weeks,and that permits a significant “de-saturation” between two successiveadministrations (successive administrations may for example be separatedby one month, two months or more). In one embodiment, a therapeuticallyactive amount of an anti-NKG2A antibody is an amount of such antibodycapable of providing (at least) the EC₅₀ concentration, optionallysubstantially the EC₁₀₀ concentration, in a tissue for NKG2A+ NK cellresponse for a period of at least 1 week, or at least about 2 weeks,following administration of the antibody, and that permits thereafter asignificant “de-saturation” between two successive administrations;optionally where the antibody is administered at a dosing frequency ofone about every month, or about once every two months. Bloodconcentration of anti-NKG2A antibody during the de-saturation period isbelow the specified concentration to be achieved during the initialperiod (e.g. 1 week, 2 weeks, etc.); for example blood and/or tissueconcentration of anti-NKG2A antibody during the de-saturation period canbe specified to be below the EC₁₀₀, optionally below the EC₅₀ for NKG2A+NK cell response.

In one embodiment, particularly where NKG2A+ T or NK cells inextravascular tissues are intended to be modulated, such as for thetreatment of a solid tumor, the anti-NKG2A antibody is administered inan amount effective to achieve a peak concentration, or to maintain ablood concentration, of about or at least about 40, 50, 60, 70 or 80μg/ml, optionally at least about 100 μg/ml, upon administration (e.g.within 1 or 2 days of administration). In one embodiment, NKG2A antibodyis administered in an amount effective to maintain the specified bloodconcentration for at least one week, optionally at least two weeks,following administration of the anti-NKG2A antibody. In one embodiment,particularly where NKG2A+ T or NK cells in extravascular tissues areintended to be modulated, such as for the treatment of a solid tumor,the anti-NKG2A antibody is administered in an amount effective toachieve, or to maintain, a continuous (minimum) blood concentration ofanti-NKG2A antibody of about or at least about 50, 60, 70 or 80 μg/ml,optionally at least about 100 μg/ml, between the first and second (andoptionally further) administrations. In one embodiment, successiveadministrations (e.g. said first and second administrations) areseparated in time by at least one week, optionally about two weeks. Theanti-NKG2A antibody can optionally be administered in an amounteffective and according to a frequency that achieves, or that maintainsa blood concentration as specified for the entire duration of theadministration cycle.

In one embodiment, provided is a method for treating or preventing asolid tumor in an individual, the method comprising administering to anindividual having a solid tumor an antibody that neutralizes theinhibitory activity of a human NKG2A polypeptide for at least oneadministration cycle, the administration cycle comprising least twoadministrations of the anti-NKG2A antibody, wherein the anti-NKG2Aantibody is administered in an amount effective to achieve, or tomaintain, a (minimum) concentration in an extravascular tissue (e.g. inthe tumor environment) of at least 4 μg/mL, optionally at least 10 μg/mLbetween two successive administrations. Optionally, the anti-NKG2Aantibody is administered in an amount effective to achieve, or tomaintain, a (minimum) concentration in an extravascular tissue (e.g. inthe tumor environment) of at least 4 μg/mL, optionally at least 10μg/mL, for the entire duration of the administration cycle. In oneembodiment, the anti-NKG2A antibody is administered in an amounteffective to maintain a continuous blood concentration of anti-NKG2Aantibody of at least 40 μg/mL, optionally at least 100 μg/mL, betweentwo successive administrations, or for the duration of theadministration cycle. In one embodiment, the anti-NKG2A antibody isadministered in an amount effective to achieve blood concentration ofanti-NKG2A antibody of at least 40 μg/mL, optionally at least 100 μg/mL,upon administration (e.g. for at least one week, at least two weeks uponadministration), followed by a period that permits a significant“de-saturation” of NKG2A-expressing cells in circulation between twosuccessive administrations (wherein blood concentration of anti-NKG2Aantibody is below said least 40 μg/mL or at least 100 μg/mL during thede-saturation period).

In one embodiment, provided is a method for treating or preventing ahematological tumor in an individual, the method comprisingadministering to an individual having a hematological tumor an antibodythat neutralizes the inhibitory activity of a human NKG2A polypeptidefor at least one administration cycle, the administration cyclecomprising at least two administrations of the anti-NKG2A antibody,wherein the anti-NKG2A antibody is administered in an amount effectiveto achieve or maintain a continuous (minimum) blood concentration ofanti-NKG2A antibody of at least 10 μg/mL between two successiveadministrations. Optionally, the anti-NKG2A antibody is administered inan amount effective to achieve or maintain a continuous (minimum) bloodconcentration of anti-NKG2A antibody of at least 10 μg/mL, for theentire duration of the administration cycle.

In one embodiment, the antibody is administered to achieve a peak bloodconcentration of at least about 40 μg/mL, optionally about or at leastabout 100 μg/mL upon administration (e.g. the day of administration,within 24 or 48 hours of administration).

In one embodiment, a blood concentration of 40 μg/ml is capable ofproviding the EC₅₀ concentration for NKG2A+ NK cell response in anextravascular tissue. In one embodiment, a blood concentration of 100μg/ml is capable of providing the EC₁₀₀ concentration in a tissue(outside of the vasculature, e.g. in the tumor environment) for NKG2A+NK cell response.

In one embodiment, a method of treatment comprises administering atreatment cycle with (a) an induction (or loading) period wherein afirst dosage of anti-NKG2A antibody is administered to achieve ormaintain a continuous(minimum) blood concentration of at least about 40μg/mL, optionally at least about 100 μg/mL, between the firstadministration and a subsequent administration of anti-NKG2A antibody,(b) a maintenance period, wherein a second, lower, dosage of anti-NKG2Aantibody is administered is an amount sufficient to achieve or maintaina continuous (minimum) blood concentration of the anti-NKG2A antibody ofat least about 40 μg/mL, optionally at least about 100 μg/mL, until thenext administrations of anti-NKG2A antibody (e.g., for the entiretreatment cycle). The maintenance period follows the induction period.In one embodiment, the maintenance period comprises at least twoadministrations of the anti-NKG2A antibody. In one embodiment, themaintenance period comprises at least two administrations at a dose andfrequency that provides a continuous blood concentration of theanti-NKG2A antibody of at least about 40 μg/mL, optionally at leastabout 100 μg/mL, between two administrations.

In any embodiment, blood concentration can be specified to be bloodserum concentration.

In one embodiment, the anti-NKG2A antibody substantially fullyneutralizes the inhibitory activity of human CD94/NKG2A in the humanpatient (in vivo), on NKG2A-positive lymphocytes in circulation or inextravascular tissue, for about one week or for about two weeks.

In one embodiment, provided is a method of treating an individual havinga cancer (e.g. a solid tumor), the method comprising administering tothe individual an antibody that binds and neutralizes the inhibitoryactivity of NKG2A for at least one administration cycle in which theanti-NKG2A antibody is administered two times per month intravenously ata dose of between 4-10 mg/kg, optionally 4-6 mg/kg, optionally 4-8mg/kg, optionally about 4 mg/kg, optionally about 6 mg/kg, optionallyabout 8 mg/kg, or optionally about 10 mg/kg body weight.

In one embodiment, provided is a method of treating an individual havinga cancer (e.g. a solid tumor), the method comprising administering tothe individual an antibody that binds and neutralizes the inhibitoryactivity of NKG2A for at least one administration cycle, wherein themethod comprises:

a. a loading period in which antibody is administered intravenously atleast once at an initial dose of between 8-10 mg/kg, optionally about 10mg/kg, and

b. a maintenance period in which the antibody is administeredintravenously every two weeks at least twice in a dose of between 2-6mg/kg, optionally between 2-5 mg/kg, optionally between 2-4 mg/kg,optionally about 2 mg/kg, optionally about 3 mg/kg, optionally about 4mg/kg, or optionally about 6 mg/kg body weight, optionally wherein thefirst administration within the maintenance period occurs no more thantwo weeks after the initial dose.

In one embodiment, a therapeutic regimen described herein isadministered to an individual having a cancer prior to the individualreceiving surgery to remove cancer cells, i.e. the anti-NKG2A antibodyregimen is used as a preoperative treatment.

In one embodiment, a therapeutic regimen or course of therapy designedto achieve a concentration of anti-NKG2A antibody that corresponds to atleast the EC₅₀, optionally the EC₁₀₀, for NKG2A+ NK cell response in theextravascular tumor environment is administered to an individual havinga cancer prior to surgery to remove cancer cells, i.e. as a preoperativetreatment.

In one embodiment, the anti-NKG2A antibody is administered to anindividual having cancer cells that express HLA-E at their surface.Optionally, HLA-E status of a cancer can be assessed prior to treatmentwith an anti-NKG2A antibody. In one embodiment provided is a methodcombining a HLA-E detection step to identify patients having HLA-E+tumor; these patients can thereafter be treated with an anti-NKG2Aantibody according to the treatment methods described herein.

In one embodiment of any of the therapeutic uses or cancer treatment orprevention methods herein, the treatment or prevention of a cancer in anindividual comprises:

a) determining the HLA-E polypeptide status of malignant cells withinthe individual having a cancer, and

b) upon a determination that the patient has HLA-E polypeptidesprominently expressed on the surface of malignant cells, administeringto the individual an anti-NKG2A antibody that binds and neutralizes theinhibitory activity of NKG2A, e.g. according to any of the treatmentmethods described herein. Optionally, the antibody interferes with thebinding of NKG2A by HLA-E.

In one embodiment of any of the therapeutic uses or cancer treatment orprevention methods herein, the treatment or prevention of a cancer in anindividual comprises:

a) determining the level of expression of HLA-E nucleic acid orpolypeptides of malignant cells within the individual having a cancer,and

b) upon a determination that malignant cells express HLA-E nucleic acidor polypeptide at a level that is increased (e.g. a high value, strongsurface staining, etc.) optionally compared to a reference level,administering to the individual an anti-NKG2A antibody that binds andneutralizes the inhibitory activity of NKG2A, e.g. according to any ofthe treatment methods described herein. Optionally, the antibodyinterferes with the binding of NKG2A by HLA-E.

In one embodiment of any of the methods, determining the HLA-Epolypeptide status or determining the level of expression in step (a)comprises determining the level of expression of a HLA-E nucleic acid orpolypeptide of malignant cells in a biological sample and comparing thelevel to a reference level (e.g. a value, weak cell surface staining,etc.). The reference level may, for example, correspond to a healthyindividual, to an individual deriving no/low clinical benefit fromtreatment with an anti-NKG2A antibody, or to an individual derivingsubstantial clinical benefit from treatment with an anti-NKG2A antibody.A determination that a biological sample expresses HLA-E nucleic acid orpolypeptide at a level that is increased (e.g. a high value, strongsurface staining, a level that corresponds to that of an individualderiving substantial clinical benefit from treatment with an anti-NKG2Aantibody, a level that is higher than that corresponding to anindividual deriving no/low clinical benefit from treatment with ananti-NKG2A antibody, etc.) indicates that the individual has a cancerthat can be treated with an anti-NKG2A antibody, e.g. according to thetreatment methods described herein.

In one embodiment, the anti-NKG2A antibody is administered as singleagent therapy. In one embodiment, the anti-NKG2A antibody isadministered in combination treatment with one or more other anti-canceragents.

In one embodiment of any of the therapeutic uses or treatment orprevention methods herein, the method further comprises administering toan individual a therapeutically active amount of a second anti-canceragent. In one embodiment, the cancer is a solid tumor. In oneembodiment, the second anti-cancer agent is an antibody that is capableof mediating ADCC (e.g. binds, via its Fc domain to human Fcγ receptors,such as CD16. In one embodiment, the antibody that mediates ADCC isadministered in an effective amount that elicits antibody-dependentcellular cytotoxicity toward human tumor cells in the human patient (invivo) that express a polypeptide to which the second anti-cancer agentis directed.

In one embodiment, the second anti-cancer agent is an anti-EGFRantibody. In one embodiment, a patient treated with an anti-NKG2Aantibody according to a treatment regimen of the invention, incombination with an anti-EGFR antibody, has an insufficient response toprior treatment with anti-EGFR antibody (e.g. is a non-responder or hasprogressing disease), or has an unfavorable prognosis for response totreatment with an anti-EGFR antibody (in the absence of treatment withanti-NKG2A).

In one aspect, the combination is administered (or is foradministration) according to a particular clinical dosage regimen,notably at a particular dose amount and according to a specific dosingschedule (e.g. a dose amount and/or according to a specific dosingschedule provided herein).

The antibody that neutralizes the inhibitory activity of a NKG2Apolypeptide (anti-NKG2A agent) is an antibody that increases the abilityof an NKG2A-expressing NK and/or T cells to cause the death of theHLA-E-expressing cell.

In one embodiment, the anti-NKG2A agent reduces the inhibitory activityof NKG2A by blocking binding of its ligand, HLA-E, i.e., the anti-NKG2Aagent interferes with the binding of NKG2A by HLA-E. The antibody havingthe heavy chain of any one of SEQ ID NOS: 2 to 6 and the light chain ofSEQ ID NO: 7 respectively, is an example of such an antibody.

In one embodiment, the anti-NKG2A antibody is antibody which binds witha significantly higher affinity to NKG2A than to one or more activatingNKG2 receptors. For example, in one embodiment, the antibody binds witha significantly higher affinity to NKG2A than to NKG2C. In an additionalor alternative embodiment, the antibody binds with a significantlyhigher affinity to NKG2A than to NKG2E. In an additional or alternativeembodiment, the antibody binds with a significantly higher affinity toNKG2A than to NKG2H. The antibody having a heavy chain of any one of SEQID NOS: 2-6 and a light chain of SEQ ID NO: 7, binds NKG2A withoutsubstantially binding to NKG2C, NKG2E or NKG2H.

In an additional or alternative embodiment, the anti-NKG2A antibodybinds the same epitope on NKG2A and/or competes for binding toCD94/NKG2A with the antibody having a heavy chain of any one of SEQ IDNOS: 2-6 and a light chain of SEQ ID NO: 7. The antibody can be, e.g., ahuman or humanized anti-NKG2A antibody.

In one embodiment, the anti-NKG2A antibody is a humanized antibodyhaving the heavy chain CDRs of any one of the heavy chain sequences ofSEQ ID NOS: 2-6 and the light chain CDRs of the light chain sequence ofSEQ ID NO: 7. Exemplary complementarity-determining region (CDR)residues or sequences and/or sites for amino acid substitutions inframework region (FR) of such humanized antibodies having improvedproperties such as, e.g., lower immunogenicity, improved antigen-bindingor other functional properties, and/or improved physicochemicalproperties such as, e.g., better stability, are provided.

In other embodiments, pharmaceutical compositions and kits are provided,as well as methods for using them.

These aspects are more fully described in, and additional aspects,features, and advantages will be apparent from, the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows receptor occupancy in human blood of anti-NKG2A antibody(humZ270 also referred to as IPH2201) at different concentrations(ng/ml) listed on the x-axis and MESF signal for receptor binding isshown on the y-axis. The antibody had a binding affinity (EC50) of about4 ng/mL for NKG2A+ cells (K_(D)˜4 ng/mL). This K_(D) is consistent withK_(D) values observed in other assays, notably affinity for binding toPBMC and affinity for recombinant NKG2A in Biacore assays. The K_(D) forfull receptor occupancy (the EC100) was about 100 ng/ml.

FIG. 2 shows predicted receptor occupancy of anti-NKG2A antibody basedon human patients treated with a single dose of HumZ270 in a 92-patientPhase I trial. Substantially complete (at least 90%) receptor saturationof NKG2A+ cells can be achieved by administering 0.03 mg/kg every twoweeks. Receptor saturation is greater with increasing doses, the plottedline in the figure having lowest receptor saturation corresponds to thelowest dose (0.01 mg/kg); each incremental higher dose level correspondsto the plotted line having the next higher receptor saturation, the linecorresponding to the highest dose (10 mg/kg) has the highest receptorsaturation. A dose of 0.1 mg/kg can be administered every four weeks tomaintain substantially complete saturation of NKG2A.

FIG. 3A shows results from an autologous cellular assay, showing thatthe concentrations of anti-NKG2A required for efficacy (NKG2A blockadein the presence of cells expressing its HLA-E ligand) are 100-foldhigher than concentrations providing full receptor saturation. The EC50concentration (the amount that provides a 50% of maximum) in the CD107mobilization assay was determined to be about 400 ng/ml, and the EC100was about 10,000 ng/ml (10 μg/ml).

FIG. 3B shows the predicted IPH2201 (anti-NKG2A) blood concentrationfollowing two-weekly i.v. injections, based on preliminary PK data andNCA analysis from a IPH2201 phase I clinical trial. Different bloodconcentrations reached with different dosages administered by i.v. everytwo weeks. IPH2201 concentration is greater with increasing doses, theplotted line in the figure having lowest blood concentration correspondsto the lowest dose (0.01 mg/kg); each incremental higher dose levelcorresponds to the plotted line provides the next higher bloodconcentration, the line corresponding to the highest dose (10 mg/kg)provides the highest blood concentration. The dose of 4 mg/kg providedan initial (peak) blood concentration of about 100 μg/ml, i.e. at aboutthe EC₁₀₀ for efficacy in tissues, and a continued (minimum) bloodconcentration above 30 μg/ml up to the two week time point, or, as ofthe fourth dose a continued (minimum) blood concentration ofapproximately 100 μg/ml. The dose of 10 mg/kg provided a continued(minimum) blood concentration approximately 100 μg/ml.

FIG. 3C shows predicted IPH2201 blood concentration following two-weeklyi.v. injections with a loading dose of 10 mg/kg body weight followed bya maintenance dose of 4 mg/kg body weight (shown as the upper line),providing an initial and continued blood concentration approximately 100μg/ml. For purposes of comparison, a constant dose of 4 mg/kg is shown(the lower line in the figure) which provides for a continued (orminimal) blood concentration of at least 30 μg/ml at two weeks.

FIGS. 4A and 4B shows ability of anti-NKG2A to enhance recognition ofHNSCC cell lines by NK cells. CD107 (Top) and CD137 (Bottom) FACSread-outs on NKG2A-NK (left) or NKG2A+ NK cells (right) are indicated,in presence of indicated target HNSCC cell lines and in presence or notof anti-NKG2A at a concentration of 10 μg/mL. The cell lines are orderedfrom left to right according to level of HLA-E surface expression. Eachdot represents PBMC from a healthy volunteer. FIG. 4A shows controls andK562 targets with low or high levels of surface HLA-E, and FIG. 4Bdemonstrates anti-NKG2A can restore lysis of HNSCC with endogenous HLA-Eexpression. This effect is only seen on NKG2A positive NK cells and isdependent on the level of expression of HLA-E.

FIG. 5 shows optimal doses of anti-NKG2A enhanced ADCC by NK cellstowards HNSCC FaDu cells induced by suboptimal doses of anti-EGFR,cetuximab (ctx).

FIGS. 6A and 6B shows effect of increasing doses of anti-NKG2A andincreasing doses of anti-EGFR (cetuximab). FIG. 3A shows CD107 read outon controls with no target and with K562-HLA-E transfectants. Eachhealthy volunteer is represented by a different symbol: squares orcircles. Crossed open symbols correspond to condition where anti-NKG2Awas replaced by 10 μg/mL hIgG4 isotypic control co-incubated with 0.1μg/mL cetuximab. FIG. 6B shows CD107 read out on HNSCC cell lines. Foreach concentration of cetuximab, the symbols (squares of circles) foreach concentration of anti-NKG2A correspond, from left to right, to 0μg/ml, 0.1 μg/ml, 1 μg/ml, and 10 μg/ml.

DEFINITIONS

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of” or by “consisting of”.

NKG2A (OMIM 161555, the entire disclosure of which is hereinincorporated by reference) is a member of the NKG2 group of transcripts(Houchins, et al. (1991) J. Exp. Med. 173:1017-1020). NKG2A is encodedby 7 exons spanning 25 kb, showing some differential splicing. Togetherwith CD94, NKG2A forms the heterodimeric inhibitory receptor CD94/NKG2A,found on the surface of subsets of NK cells, α/β T cells, γ/δ T cells,and NKT cells. Similar to inhibitory KIR receptors, it possesses an ITIMin its cytoplasmic domain. As used herein, “NKG2A” refers to anyvariant, derivative, or isoform of the NKG2A gene or encoded protein.Also encompassed are any nucleic acid or protein sequences sharing oneor more biological properties or functions with wild type, full lengthNKG2A, and sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, orhigher nucleotide or amino acid identity. Human NKG2A comprises 233amino acids in 3 domains, with a cytoplasmic domain comprising residues1-70, a transmembrane region comprising residues 71-93, and anextracellular region comprising residues 94-233, of the followingsequence:

(SEQ ID NO: 1) MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGND-KTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGH-CPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSS-WIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL.

NKG2C (OMIM 602891, the entire disclosure of which is hereinincorporated by reference) and NKG2E (OMIM 602892, the entire disclosureof which is herein incorporated by reference) are two other members ofthe NKG2 group of transcripts (Gilenke, et al. (1998) Immunogenetics48:163-173). The CD94/NKG2C and CD94/NKG2E receptors are activatingreceptors found on the surface of subsets of lymphocytes such as NKcells and T-cells.

HLA-E (OMIM 143010, the entire disclosure of which is hereinincorporated by reference) is a nonclassical MHC molecule that isexpressed on the cell surface and regulated by the binding of peptides,e.g., such as fragments derived from the signal sequence of other MHCclass I molecules. Soluble versions of HLA-E have also been identified.In addition to its T-cell receptor binding properties, HLA-E bindssubsets of natural killer (NK) cells, natural killer T-cells (NKT) and Tcells (α/β and γ/δ), by binding specifically to CD94/NKG2A, CD94/NKG2B,and CD94/NKG2C (see, e.g., Braud et al. (1998) Nature 391:795-799, theentire disclosure of which is herein incorporated by reference). Surfaceexpression of HLA-E protects target cells from lysis by CD94/NKG2A+ NK,T, or NKT cell clones. As used herein, “HLA-E” refers to any variant,derivative, or isoform of the HLA-E gene or encoded protein. Alsoencompassed are any nucleic acid or protein sequences sharing one ormore biological properties or functions with wild type, full lengthHLA-E, and sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, orhigher nucleotide or amino acid identity.

In the context of the present invention, “CD94/NKG2A positivelymphocyte” refers to cells of the lymphoid lineage (e.g. NK-, NKT- andT-cells) expressing CD94/NKG2A on the cell-surface, which can bedetected by e.g. flow-cytometry using antibodies that specificallyrecognize a combined epitope on CD94 and NKG2A or and epitope on NKG2Aalone. “CD94/NKG2A positive lymphocyte” also includes immortal celllines of lymphoid origin (e.g. NKL, NK-92).

In the context of the present invention, “reduces the inhibitoryactivity of NKG2A”, “neutralizes NKG2A” or “neutralizes the inhibitoryactivity of NKG2A” refers to a process in which CD94/NKG2A is inhibitedin its capacity to negatively affect intracellular processes leading tolymphocyte responses such as cytokine release and cytotoxic responses.This can be measured for example in a NK- or T-cell based cytotoxicityassay, in which the capacity of a therapeutic compound to stimulatekilling of HLA-E positive cells by CD94/NKG2A positive lymphocytes ismeasured. In one embodiment, an antibody preparation causes at least a10% augmentation in the cytotoxicity of a CD94/NKG2A-restrictedlymphocyte, preferably at least a 40% or 50% augmentation in lymphocytecytotoxicity, or more preferably at least a 70% augmentation in NKcytotoxicity”, and referring to the cytotoxicity assays described. If ananti-NKG2A antibody reduces or blocks CD94/NKG2A interactions withHLA-E, it may increase the cytotoxicity of CD94/NKG2A-restrictedlymphocytes. This can be evaluated, for example, in a standard 4-hour invitro cytotoxicity assay using, e.g., NK cells that express CD94/NKG2A,and target cells that express HLA-E. Such NK cells do not efficientlykill targets that express HLA-E because CD94/NKG2A recognizes HLA-E,leading to initiation and propagation of inhibitory signaling thatprevents lymphocyte-mediated cytolysis. Such an in vitro cytotoxicityassay can be carried out by standard methods that are well known in theart, as described for example in Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993). Chromium release and/or other parameters to assess theability of the antibody to stimulate lymphocytes to kill target cellssuch as P815, K562 cells, or appropriate tumor cells are also disclosedin Sivori et al., J. Exp. Med. 1997; 186:1129-1136; Vitale et al., J.Exp. Med. 1998; 187:2065-2072; Pessino et al. J. Exp. Med. 1998;188:953-960; Neri et al. Clin. Diag. Lab. Immun. 2001; 8:1131-1135;Pende et al. J. Exp. Med. 1999; 190:1505-1516, the entire disclosures ofeach of which are herein incorporated by reference. The target cells arelabeled with ⁵¹Cr prior to addition of NK cells, and then the killing isestimated as proportional to the release of ⁵¹Cr from the cells to themedium, as a result of killing. The addition of an antibody thatprevents CD94/NKG2A from binding to HLA-E results in prevention of theinitiation and propagation of inhibitory signaling via CD94/NKG2A.Therefore, addition of such agents results in increases inlymphocyte-mediated killing of the target cells. This step therebyidentifies agents that prevent CD94/NKG2A-induced negative signaling by,e.g., blocking ligand binding. In a particular ⁵¹Cr-release cytotoxicityassay, CD94/NKG2A-expressing NK effector-cells can kill HLA-E-negativeLCL 721.221 target cells, but less well HLA-E-expressing LCL 721.221-Cw3control cells. In contrast, YTS effector-cells that lack CD94/NKG2A killboth cell-lines efficiently. Thus, NK effector cells kill lessefficiently HLA-E⁺ LCL 721.221-Cw3 cells due to HLA-E-induced inhibitorysignaling via CD94/NKG2A. When NK cells are pre-incubated with blockinganti-CD94/NKG2A antibodies according to the present invention in such a⁵¹Cr-release cytotoxicity assay, HLA-E-expressing LCL 721.221-Cw3 cellsare more efficiently killed, in an antibody-concentration-dependentfashion. The inhibitory activity (i.e. cytotoxicity enhancing potential)of an anti-NKG2A antibody can also be assessed in any of a number ofother ways, e.g., by its effect on intracellular free calcium asdescribed, e.g., in Sivori et al., J. Exp. Med. 1997; 186:1129-1136, thedisclosure of which is herein incorporated by reference. Activation ofNK cell cytotoxicity can be assessed for example by measuring anincrease in cytokine production (e.g. IFN-γ production) or cytotoxicitymarkers (e.g. CD107 or CD137 mobilization). In an exemplary protocol,IFN-γ production from PBMC is assessed by cell surface andintracytoplasmic staining and analysis by flow cytometry after 4 days inculture. Briefly, Brefeldin A (Sigma Aldrich) is added at a finalconcentration of 5 μg/ml for the last 4 hours of culture. The cells arethen incubated with anti-CD3 and anti-CD56 mAb prior to permeabilization(IntraPrep™; Beckman Coulter) and staining with PE-anti-IFN-γ or PE-IgG1(Pharmingen). GM-CSF and IFN-γ production from polyclonal activated NKcells are measured in supernatants using ELISA (GM-CSF: DuoSet Elisa,R&D Systems, Minneapolis, Minn., IFN-γ: OptEIA set, Pharmingen).

Whenever within this whole specification “treatment of cancer” or thelike is mentioned with reference to anti-NKG2A binding agent (e.g.antibody), there is meant: (a) method of treatment of cancer, saidmethod comprising the step of administering (for at least one treatment)an anti-NKG2A binding agent, (preferably in a pharmaceuticallyacceptable carrier material) to an individual, a mammal, especially ahuman, in need of such treatment, in a dose that allows for thetreatment of cancer, (a therapeutically effective amount), preferably ina dose (amount) as specified herein; (b) the use of an anti-NKG2Abinding agent for the treatment of cancer, or an anti-NKG2A bindingagent, for use in said treatment (especially in a human); (c) the use ofan anti-NKG2A binding agent for the manufacture of a pharmaceuticalpreparation for the treatment of cancer, a method of using an anti-NKG2Abinding agent for the manufacture of a pharmaceutical preparation forthe treatment of cancer, comprising admixing an anti-NKG2A binding agentwith a pharmaceutically acceptable carrier, or a pharmaceuticalpreparation comprising an effective dose of an anti-NKG2A binding agentthat is appropriate for the treatment of cancer; or (d) any combinationof a), b), and c), in accordance with the subject matter allowable forpatenting in a country where this application is filed.

The term “biopsy” as used herein is defined as removal of a tissue forthe purpose of examination, such as to establish diagnosis. Examples oftypes of biopsies include by application of suction, such as through aneedle attached to a syringe; by instrumental removal of a fragment oftissue; by removal with appropriate instruments through an endoscope; bysurgical excision, such as of the whole lesion; and the like.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG are the exemplary classes of antibodies employedherein because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Optionally the antibody is a monoclonal antibody. Particular examples ofantibodies are humanized, chimeric, human, or otherwise-human-suitableantibodies. “Antibodies” also includes any fragment or derivative of anyof the herein described antibodies.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.NKG2A, as assessed using either recombinant forms of the proteins,epitopes therein, or native proteins present on the surface of isolatedtarget cells. Competitive binding assays and other methods fordetermining specific binding are well known in the art. For examplebinding can be detected via radiolabels, physical methods such as massspectrometry, or direct or indirect fluorescent labels detected using,e.g., cytofluorometric analysis (e.g. FACScan). Binding above the amountseen with a control, non-specific agent indicates that the agent bindsto the target. An agent that specifically binds NKG2A may bind NKG2Aalone or NKG2A as a dimer with CD94.

When an antibody is said to “compete with” a particular monoclonalantibody, it means that the antibody competes with the monoclonalantibody in a binding assay using either recombinant molecules (e.g.,NKG2A) or surface expressed molecules (e.g., NKG2A). For example, if atest antibody reduces the binding of an antibody having a heavy chain ofany one of SEQ ID NOS: 2-6 and a light chain of SEQ ID NO: 7 to a NKG2Apolypeptide or NKG2A-expressing cell in a binding assay, the antibody issaid to “compete” respectively with such antibody.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab−Ag], where[Ab−Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One standard method well known in the art for determining theaffinity of mAbs is the use of surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device).

Within the context herein a “determinant” designates a site ofinteraction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody binds. A protein epitopemay comprise amino acid residues directly involved in the binding aswell as amino acid residues which are effectively blocked by thespecific antigen binding antibody or peptide, i.e., amino acid residueswithin the “footprint” of the antibody. It is the simplest form orsmallest structural area on a complex antigen molecule that can combinewith e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “therapeutic agent” refers to anagent that has biological activity.

For the purposes herein, a “humanized” or “human” antibody refers to anantibody in which the constant and variable framework region of one ormore human immunoglobulins is fused with the binding region, e.g. theCDR, of an animal immunoglobulin. Such antibodies are designed tomaintain the binding specificity of the non-human antibody from whichthe binding regions are derived, but to avoid an immune reaction againstthe non-human antibody. Such antibodies can be obtained from transgenicmice or other animals that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge (see, e.g., Green etal. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856;Taylor et al. (1994) Int Immun 6:579, the entire teachings of which areherein incorporated by reference). A fully human antibody also can beconstructed by genetic or chromosomal transfection methods, as well asphage display technology, all of which are known in the art (see, e.g.,McCafferty et al. (1990) Nature 348:552-553). Human antibodies may alsobe generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos.5,567,610 and 5,229,275, which are incorporated in their entirety byreference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ε and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, Md.).

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

Within the context herein, the term antibody that “binds” a polypeptideor epitope designates an antibody that binds said determinant withspecificity and/or affinity.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Computer programmethods for determining identity between two sequences include the GCGprogram package, including GAP (Devereux et al., Nucl. Acid. Res. 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison,Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215,403-410 (1990)). The BLASTX program is publicly available from theNational Center for Biotechnology Information (NCBI) and other sources(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschulet al., supra). The well-known Smith Waterman algorithm may also be usedto determine identity.

Production of Antibodies

The anti-NKG2A agent binds an extra-cellular portion of human CD94/NKG2Areceptor and reduces the inhibitory activity of human CD94/NKG2Areceptor expressed on the surface of a CD94/NKG2A positive lymphocyte.In one embodiment the agent competes with HLA-E in binding toCD94/NKG2A, i.e. the agent interferes with and reduces the interactionbetween CD94/NKG2A and its ligand HLA-E. The antibody may bind acombined epitope on CD94 and NKG2A or an epitope on NKG2A alone. In oneembodiment, the antibody binds an epitope on NKG2A which at least partlyoverlaps with the HLA-E binding site.

In one aspect the anti-NKG2A agent is an antibody selected from a fullyhuman antibody, a humanized antibody, and a chimeric antibody. In oneaspect, the agent comprises a constant domain derived from a human IgG1,IgG2, IgG3 or IgG4 antibody. In one aspect, the agent is a fragment ofan antibody selected from IgA, an IgD, an IgG, an IgE and an IgMantibody. In one aspect, the agent is an antibody fragment selected froma Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab)2 fragment,a F(ab′)2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camelIg), a V_(HH) fragment, a single domain FV, and a single-chain antibodyfragment. In one aspect, the agent is a synthetic or semisyntheticantibody-derived molecule selected from a scFV, a dsFV, a minibody, adiabody, a triabody, a kappa body, an IgNAR; and a multispecificantibody.

Preferably, the anti-NKG2A antibodies do not demonstrate substantialspecific binding to Fcγ receptors, e.g. CD16. Such antibodies maycomprise constant regions of various heavy chains that are known not tobind Fc receptors. One such example is a human IgG4 constant region. Inone embodiment, the IgG4 antibody comprises a modification to preventthe formation of half antibodies (fab arm exchange) in vivo, e.g., theantibody comprises an IgG4 heavy chain comprising a serine to prolinemutation in residue 241, corresponding to position 228 according to theEU-index (Kabat et al., “Sequences of proteins of immunologicalinterest”, 5^(th) ed., NIH, Bethesda, M L, 1991). Such modified IgG4antibodies will remain intact in vivo and maintain a bivalent (highaffinity) binding to NKG2A, as opposed to native IgG4 that will undergofab arm exchange in vivo such that they bind to NKG2A in monovalentmanner which can alter binding affinity. Alternatively, antibodyfragments that do not comprise one or more constant regions, such as Fabor F(ab′)2 fragments, can be used to avoid Fc receptor binding. Fcreceptor binding can be assessed according to methods known in the art,including for example testing binding of an antibody to Fc receptorprotein in a BIACORE assay. Also, any human antibody type (e.g. IgG1,IgG2, IgG3 or IgG4) can be used in which the Fc portion is modified tominimize or eliminate binding to Fc receptors (see, e.g., WO03101485,the disclosure of which is herein incorporated by reference). Assayssuch as, e.g., cell based assays, to assess Fc receptor binding are wellknown in the art, and are described in, e.g., WO03101485.

An anti-NKG2A antibody can advantageously bind to an extracellularportion of NKG2A with a KD that is at least 100 fold lower than the KDfor binding to NKG2C. In a one aspect, the antibody binds to anextracellular portion of NKG2A with a KD that is at least 150, 200, 300,400, or 10,000 fold lower than the KD for binding to NKG2C. In anotheraspect, the antibody binds to an extracellular portion of NKG2A with aKD that is at least 100 fold lower than the KD for binding to NKG2C,NKG2E and/or NKG2H molecules. In a further aspect, the antibody binds toan extracellular portion of NKG2A with a KD that is at least 150, 200,300, 400, or 10,000 fold lower than the KD for binding to NKG2C, NKG2Cand/or NKG2H molecules. This can be measured, for instance, in BiaCoreexperiments, in which the capacity of agents to bind the extracellularportion of immobilized CD94/NKG2A (e.g. purified from CD94/NKG2expressing cells, or produced in a bio-system) is measured and comparedto the binding of agents to similarly produced CD94/NKG2C and/or otherCD94/NKG2 variants in the same assay. Alternatively, the binding ofantibodies to cells that either naturally express, or over-express (e.g.after transient or stable transfection), CD94/NKG2A can be measured andcompared to binding of cells expressing CD94/NKG2C and/or otherCD94/NKG2 variants. Anti-NKG2A antibodies may optionally bind NKG2B,which is an NKG2A splice variant forming an inhibitory receptor togetherwith CD94. In one embodiment, affinity can be measured using the methodsdisclosed in U.S. Pat. No. 8,206,709, for example by assessing bindingto covalently immobilized NKG2A-CD94-Fc fusion protein by Biacore asshown in Example 8 of U.S. Pat. No. 8,206,709, the disclosure of whichis incorporate herein by reference.

The antibody can for example have an EC₅₀ for binding (high affinity) toNKG2A-expressing cells of between 0.5-10 ng/ml, optionally 1-5 ng/ml,optionally 1-10 ng/ml, optionally 1-20 ng/ml, e.g. about 4 ng/ml. TheNKG2A-expressing cells can be, for example, NKG2A-expressing cells inhuman PBMC. In one embodiment, the NKG2A-expressing cells are cells madeto express CD94/NKG2A, for example Ba/F3 cells stably overexpressingCD94/NKG2A as shown in Example 13 of U.S. Pat. No. 8,206,709, thedisclosure of which is incorporated by reference. In one embodiment, theantibody has binding affinity (K_(D)), optionally wherein bindingaffinity is bivalent, for a human NKG2A polypeptide of less than 10⁻⁹ M,optionally less than 10⁻¹⁰ M, or optionally less than 10⁻¹¹M, optionallybetween than 10⁻¹⁰ M and 10⁻¹²M, optionally between than 10⁻¹⁰ M and10⁻¹¹M. Affinity can be assessed, for example, for binding to asingle-chain NKG2A-CD94-mFc construct as described in U.S. Pat. No.7,932,055, the disclosure of which is incorporated by reference).

The anti-NKG2A antibody can be a human or humanized antibody, forexample comprising a VH human acceptor framework from a human acceptorsequence selected from, e.g., VH1_18, VH5_a, VH5_51, VH1_f, and VH1_46,and a JH6 J-segment, or other human germline VH framework sequencesknown in the art. The VL region human acceptor sequence may be, e.g.,VKI_O2/JK4.

In one embodiment, the antibody is a humanized antibody based onantibody Z270. Different humanized Z270VH chains are shown in SEQ IDNOS: 2-6 (variable region domain amino acids underlined). HumanizedZ270VH light chain is shown in SEQ ID NO: 7. HumZ270 antibody is alsodisclosed in U.S. Pat. No. 8,206,709 (the disclosure of which isincorporated herein by reference). HumZ270VH6 (SEQ ID NO: 2) is based onVH5_51; HumZ270VH1 (SEQ ID NO: 3) is based on VH1_18; humZ270VH5 (SEQ IDNO: 4) is based on VH5_a; humZ270VH7 (SEQ ID NO: 5) is based on VH1_f;and humZ270VH8 (SEQ ID NO: 6) is based on VH1_46; all with a JH6J-segment. Each of these antibodies retains high affinity binding toNKG2A, with low likelihood of a host immune response against theantibody as the 6 C-terminal amino acid residues of the Kabat CDR-H2 ofeach of the humanized constructs are identical to the human acceptorframework. Using the alignment program VectorNTI, the following sequenceidentities between humZ270VH1 and humZ270VH5, -6, -7, and -8 wereobtained: 78.2% (VH1 vs. VH5), 79.0% (VH1 vs. VH6), 88.7% (VH1 vs. VH7),and 96.0% (VH1 vs. VH8).

In one aspect, the agent comprises (i) a heavy chain variable region ofany of SEQ ID NOS: 2-6, or an amino acid sequence at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) a light chainvariable region of SEQ ID NO: 7, or an amino acid sequence at least 50%,60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto. In one aspect,the agent comprises (i) a heavy chain comprising the amino acid sequenceof any of SEQ ID NOS: 2-6, or an amino acid sequence at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) a light chaincomprising the amino acid sequence of SEQ ID NO: 7, or an amino acidsequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identicalthereto. The antibody having the heavy chain comprising the sequence ofany of SEQ ID NOS: 2-6 and a light chain comprising the sequence of SEQID NO: 7 neutralizes the inhibitory activity of NKG2A, but does notsubstantially bind the activating receptors NKG2C, NKGE or NKG2H. Thisantibody furthermore competes with HLA-E for binding to NKG2A on thesurface of a cell. In one aspect, the agent comprises HCDR1, HCDR2and/or HCDR3 sequences derived from the heavy chain having the aminoacid sequence of any of SEQ ID NO: 2-6. In one aspect of the invention,the agent comprises LCDR1, LCDR2 and/or LCDR3 sequences derived from thelight chain having the amino acid sequence of SEQ ID NO: 7.

Heavy Chains (variable regions underlined) VH6: (SEQ ID NO: 2)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYD-SETHYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG-PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH1: (SEQ ID NO: 3)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYA-QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYS-LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS VH5: (SEQ ID NO: 4)EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYD-SETHYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDFDVGTLY-WFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH7: (SEQ ID NO: 5)EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMNWVQQAPGKGLEWMGRIDPYDSETHYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK VH8: (SEQ ID NO: 6)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPYDSETHYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDFDVGTLYWFFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain (SEQ ID NO: 7)DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-H1 corresponding to residues 31-35 of any of SEQ ID NOS: 2-6 (theamino acid sequence SYWMN (SEQ ID NO: 8)), a CDR-H2 corresponding toresidues 50-60 (the amino acid sequence RIDPYDSETHY (SEQ ID NO: 9))(optionally 50-66 when including the 6 terminal amino acids of humanorigin, i.e. the sequence RIDPYDSETHYSPSFQG (SEQ ID NO: 10) for the VH6heavy chain, the sequence RIDPYDSETHYAQKLQG (SEQ ID NO: 11) for the VH1heavy chain, etc.) of any of SEQ ID NOS: 2-6, and a CDR-H3 correspondingto residues 99-114 (95-102 according to Kabat) of any of SEQ ID NOS: 2-6(the amino acid sequence GGYDFDVGTLYWFFDV (SEQ ID NO: 12)). In oneembodiment, the CDR-H2 corresponding to residues 50-66 of any of SEQ IDNOS: 2-6. Optionally, a CDR may comprise one, two, three, four, or moreamino acid substitutions.

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-L1 corresponding to residues 24-34 of SEQ ID NO: 7 (the amino acidsequence RASENIYSYLA (SEQ ID NO: 13)), a CDR-L2 corresponding toresidues 50-56 of SEQ ID NO: 7 (the amino acid sequence NAKTLAE (SEQ IDNO: 14)), and an CDR-L3 corresponding to residues 89-97 of SEQ ID NO: 7(the amino acid sequence QHHYGTPRT (SEQ ID NO: 15)). Optionally, a CDRmay comprise one, two, three, four, or more amino acid substitutions.

In one aspect, the anti-NKG2A antibody is an antibody comprising aCDR-H1 corresponding to residues 31-35 of any of SEQ ID NOS: 2-6, aCDR-H2 corresponding to residues 50-60 (optionally 50-66) of any of SEQID NOS: 2-6, and a CDR-H3 corresponding to residues 99-114 (95-102according to Kabat) of any of SEQ ID NOS: 2-6, a CDR-L1 corresponding toresidues 24-34 of SEQ ID NO: 7, a CDR-L2 corresponding to residues 50-56of SEQ ID NO: 7, and an CDR-L3 corresponding to residues 89-97 of SEQ IDNO: 7.

In one aspect, the agent is a fully human antibody which has been raisedagainst the CD94/NKG2A epitope to which any of the aforementionedantibodies bind.

It will be appreciated that, while the aforementioned antibodies can beused, other antibodies can be prepared. For example, any fragment ofNKG2A, preferably but not exclusively human NKG2A, or any combination ofNKG2A fragments, can be used as immunogens to raise antibodies, and theantibodies can recognize epitopes at any location within the NKG2Apolypeptide, so long as they can do so on NKG2A expressing NK cells asdescribed herein. Most preferably, the epitope is the epitopespecifically recognized by antibody having the heavy chain of any of SEQID NOS: 2-6 and the light chain of SEQ ID NO: 7.

In one aspect, the agent competes with humZ270 antibody disclosed inU.S. Pat. No. 8,206,709 (the disclosure of which is incorporated hereinby reference) in binding to the extra-cellular portion of humanCD94/NKG2A receptor. Competitive binding can be measured, for instance,in BiaCore experiments, in which the capacity of agents is measured, forbinding the extracellular portion of immobilized CD94/NKG2A receptor(e.g. purified from CD94/NKG2 expressing cells, or produced in abio-system) saturated with humZ270. Alternatively, the binding of agentsto cells is measured that either naturally express, or over-express(e.g. after transient or stable transfection), CD94/NKG2A receptor, andwhich have been pre-incubated with saturating doses of Z270. In oneembodiment, competitive binding can be measured using the methodsdisclosed in U.S. Pat. No. 8,206,709, for example by assessing bindingto Ba/F3-CD94-NKG2A cells by flow cytometry as shown in Example 15 ofU.S. Pat. No. 8,206,709, the disclosure of which is incorporate hereinby reference.

An anti-NKG2A antibody can be incorporated in a pharmaceuticalformulation comprising in a concentration from 1 mg/ml to 500 mg/ml,wherein said formulation has a pH from 2.0 to 10.0. The formulation mayfurther comprise a buffer system, preservative(s), tonicity agent(s),chelating agent(s), stabilizers and surfactants. In one embodiment, thepharmaceutical formulation is an aqueous formulation, i.e., formulationcomprising water. Such formulation is typically a solution or asuspension. In a further embodiment, the pharmaceutical formulation isan aqueous solution. The term “aqueous formulation” is defined as aformulation comprising at least 50% w/w water. Likewise, the term“aqueous solution” is defined as a solution comprising at least 50% w/wwater, and the term “aqueous suspension” is defined as a suspensioncomprising at least 50% w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment, the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

In another embodiment, the pH of the formulation is in the rangeselected from the list consisting of from about 2.0 to about 10.0, about3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, andabout 5.5 to about 7.5.

In a further embodiment, the buffer is selected from the groupconsisting of sodium acetate, sodium carbonate, citrate, glycylglycine,histidine, glycine, lysine, arginine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment, the formulation further comprises apharmaceutically acceptable preservative. In a further embodiment, theformulation further comprises an isotonic agent. In a furtherembodiment, the formulation also comprises a chelating agent. In afurther embodiment of the invention the formulation further comprises astabilizer. In a further embodiment, the formulation further comprises asurfactant. For convenience reference is made to Remington: The Scienceand Practice of Pharmacy, 19^(th) edition, 1995.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing an antibody may be administeredto a patient in need of such treatment at several sites, for example, attopical sites, for example, skin and mucosal sites, at sites whichbypass absorption, for example, administration in an artery, in a vein,in the heart, and at sites which involve absorption, for example,administration in the skin, under the skin, in a muscle or in theabdomen. Administration of pharmaceutical compositions may be throughseveral routes of administration, for example, subcutaneous,intramuscular, intraperitoneal, intravenous, lingual, sublingual,buccal, in the mouth, oral, in the stomach and intestine, nasal,pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Diagnosis and Treatment of Malignancies

Described are methods useful in the diagnosis, prognosis, monitoring,treatment and prevention of a cancer in an individual. While the methodsdescribed herein are particularly useful for the treatment of solidtumors, the treatment regimens described herein can also be used for avariety of hematological cancers, as well as infectious disease, andinflammation and autoimmune disorders. The methods and compositions ofthe present invention are utilized for example the treatment of avariety of cancers and other proliferative diseases including, but notlimited to: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroidand skin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia,chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-celllymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma,hairy cell lymphoma and Burketts lymphoma, and multiple myeloma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias, promyelocytic leukemia, and myelodysplasticsyndrome; tumors of mesenchymal origin, including fibrosarcoma andrhabdomyoscarcoma; other tumors, including melanoma, seminoma,terato-carcinoma, neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma.

Examples of hematopoietic tumors of lymphoid lineage, include forexample T-cell and B-cell tumors, including but not limited to T-celldisorders such as T-prolymphocytic leukemia (T-PLL), including of thesmall cell and cerebriform cell type; large granular lymphocyte leukemia(LGL) preferably of the T-cell type; Sezary syndrome (SS); adult T-cellleukemia lymphoma (ATLL); T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinalT-cell lymphoma; T-lymphoblastic; lymphoma/leukaemia (T-Lbly/T-ALL),multiple myeloma.

In one embodiment, the cancer is a head and neck squamous cell carcinoma(HNSCC). In one embodiment the HNSCC is an oropharangeal tumor, a larynxtumor, a tumor of the oral cavity, or a tumor of the hypopharynx. In oneembodiment, the HNSCC is an oral cavity SCC (OCSCC). OCSCC comprisessquamous cell carcinoma of the lip, anterior 2/3 of the tongue, floor ofthe mouth, buccal mucosa, gingiva, hard palate and retromolar trigone.In one embodiment the HNSCC is a metastatic cancer.

When treating an individual having a solid tumor, a compound (e.g.antibody) that neutralizes the inhibitory activity of a human NKG2Apolypeptide can advantageously be administered according to a treatmentregimen described herein, to an individual having a cancer who has notreceived surgery to remove cancer cells, or who has not in the currentperiod received such surgery. However it will be appreciated that thecompound can also be administered to a patient who has received, or whois undergoing, surgery to remove cancer cells. Where the anti-NKG2Acompound is administered to an individual who has not received surgicalintervention to remove cancer cells (e.g. to remove HNSCC cells), theNKG2A-binding compound can for example be administered approximately 1to 8 weeks prior to surgery. In one embodiment, at least one (e.g. one,two, three or more) complete administration cycle(s) of treatment withanti-NKG2A compound is administered prior to surgery. In one embodiment,the administration cycle is between 2 weeks and 8 weeks.

In one embodiment, the cancer treated with the methods disclosed hereinis an HLA-E-expressing cancer.

A patient having a cancer can be treated with the anti-NKG2A agents withor without a prior detection step to assess expression of HLA-E on thesurface of tumor cells. Advantageously, the treatment methods cancomprises a step of detecting a HLA-E nucleic acid or polypeptide in abiological sample of a tumor (e.g. on a tumor cell) from an individual.Example of biological samples include any suitable biological fluid (forexample serum, lymph, blood), cell sample, or tissue sample. Forexample, a tissue sample may be a sample of tumor tissue ortumor-adjacent tissue. Optionally, HLA-E polypeptide is detected on thesurface of a malignant cell. A determination that a biological sampleexpresses HLA-E (e.g. prominently expresses; expresses HLA-E at a highlevel, high intensity of staining with an anti-HLA-E antibody, comparedto a reference) indicates that the individual has a cancer that may havea strong benefit from treatment with an agent that inhibits NKG2A. Inone embodiment, the method comprises determining the level of expressionof a HLA-E nucleic acid or polypeptide in a biological sample andcomparing the level to a reference level (e.g. a value, weak cellsurface staining, etc.) corresponding to a healthy individual. Adetermination that a biological sample expresses an HLA-E nucleic acidor polypeptide at a level that is increased compared to the referencelevel indicates that the individual has a cancer that can be treatedwith an agent that inhibits NKG2A.

In one embodiment, a determination that a biological sample (e.g., asample comprising tumor cells, tumor tissue and/or tumor adjacenttissue) prominently expresses HLA-E nucleic acid or polypeptideindicates that the individual has a cancer that can be treated with anagent that inhibits NKG2A. “Prominently expressed”, when referring to aHLA-E polypeptide, means that the HLA-E polypeptide is expressed in asubstantial number of tumor cells taken from a given individual. Whilethe definition of the term “prominently expressed” is not bound by aprecise percentage value, in some examples a receptor said to be“prominently expressed” will be present on at least 30%, 40%, 50° %,60%, 70%, 80%, or more of the tumor cells taken from a patient.

Determining whether an individual has cancer cells that express an HLA-Epolypeptide can for example comprise obtaining a biological sample (e.g.by performing a biopsy) from the individual that comprises cancer cells,bringing said cells into contact with an antibody that binds an HLA-Epolypeptide, and detecting whether the cells express HLA-E on theirsurface. Optionally, determining whether an individual has cancer cellsthat express HLA-E comprises conducting an immunohistochemistry assay.Optionally determining whether an individual has cancer cells thatexpress HLA-E comprises conducting a flow cytometry assay.

In one exemplary aspect, provided is method of reducing progression ofcancer in a mammalian host, (e.g., a human patient) having a detectablelevel of cancer cells comprising administering an anti-NKG2A antibody ina dosage and frequency according to the disclosure sufficient todetectably reduce the progression of the cancer in the host.

In one embodiment, provided is a method for treating or preventing adisease (e.g. a hematological tumor, an inflammatory or autoimmunedisease, an infection) in an individual, the method comprisingadministering to an individual having disease an antibody thatneutralizes the inhibitory activity of a human NKG2A polypeptide in anamount that achieves a concentration in circulation that is at least 10,20, 30 or 50 times higher than the concentration required forsubstantially full (e.g., 90%, 95%) receptor saturation (e.g., asassessed by titrating anti-NKG2A antibody on NKG2A-expressing cells, forexample in PBMC). The antibody can for example have an EC₅₀ for bindingto NKG2A-expressing cells in human PBMC of between 0.5-10 ng/ml,optionally 1-10 ng/ml, optionally 1-20 ng/ml, e.g. about 4 ng/ml.

In one embodiment, provided is a method for treating or preventing adisease (e.g. a solid tumor, an inflammatory or autoimmune disease, aninvention) in an individual, the method comprising administering to anindividual having disease an antibody that neutralizes the inhibitoryactivity of a human NKG2A polypeptide in an amount that achieves aconcentration in an extravascular tissue of interest (e.g. the tumor ortumor environment) that is at least 10, 20, 30 or 50 times higher thanthe concentration required for substantially full (e.g., 90%, 95%)receptor saturation (e.g., as assessed by titrating anti-NKG2A antibodyon NKG2A-expressing cells, for example in PBMC).

The EC₅₀ for NKG2A+ NK cell response of the blocking anti-NKG2A antibodyHumZ270 is about 4 μg/ml, thus an amount of this anti-NKG2A antibody isadministered so at to achieve and/or maintain a blood concentration ofat least 4 μg/ml. Advantageously an amount of anti-NKG2A antibody isadministered to an individual so at to achieve and/or maintain a bloodconcentration in the individual of at least 10 μg/ml (the EC₁₀₀ forNKG2A+NK cell response. For example, the blood concentration to beachieved and/or maintained can be between 10-12 μg/ml, 10-15 μg/ml,10-20 μg/ml, 10-30 μg/ml, 10-40 μg/ml, 10-50 μg/ml, 10-70 μg/ml, 10-100μg/ml, 10-150 μg/ml or 10-200 μg/ml. In one embodiment, an amount ofanti-NKG2A antibody is administered to an individual so at to achieveand/or maintain a tissue concentration in the individual of at leastabout 4 μg/ml (the EC₅₀ for NKG2A+ NK cell response) or optionally atleast about 10 μg/ml (the EC₁₀₀ for NKG2A+ NK cell response). Whentissues outside of the vasculature are targeted (e.g. in the treatmentof solid tumors), an amount of anti-NKG2A antibody is administered so atto achieve and/or maintain a tissue concentration of at least 10 μg/ml;for example, administering an amount of anti-NKG2A antibody to achieve ablood concentration of at least 100 μg/ml is expected to achieve anextravascular tissue (e.g. tumor tissue) concentration of at least 10μg/ml. For example, the blood concentration to be achieved and/ormaintained in order to achieve/maintain 10 μg/ml in a tissue can bebetween 100-110 μg/ml, 100-120 μg/ml, 100-130 μg/ml, 100-140 μg/ml,100-150 μg/ml, 100-200 μg/ml, 100-250 μg/ml or 100-300 μg/ml.

In some embodiments, an amount of anti-NKG2A antibody is administered soas to obtain a concentration in blood (serum) that corresponds to atleast the EC₅₀ for NKG2A+ NK cell response, optionally at about or atleast about, the EC₁₀₀. NKG2A+ NK cell response can be assessed using asuitable assay of cytotoxic activity of NKG2A-expressing NK cells towardHLA-E expressing target cells. Examples include assays based on markersof NK cell activation, for example CD107 or CD137 expression as shown inthe Examples herein. “EC₅₀” with respect to NKG2A+ NK cell response,refers to the efficient concentration of anti-NKG2A antibody whichproduces 50% of its maximum response or effect with respect to suchNKG2A+ NK cell response. “EC₁₀₀” with respect to NKG2A+ NK cellresponse, refers to the efficient concentration of anti-NKG2A antibodywhich produces its substantially maximum response or effect with respectto such NKG2A+ NK cell response. In some embodiments, particularly forthe treatment of solid tumors, the concentration achieved is designed tolead to a concentration in tissues (outside of the vasculature, e.g. inthe tumor or tumor environment) that corresponds to at least the EC₅₀for NKG2A+ NK cell response, optionally at about, or at least about, theEC₁₀₀.

Suitable treatment protocols for treating a human include, for example,administering to the patient an effective amount of an anti-NKG2Aantibody, wherein the method comprises at least one administration cyclein which at least one dose of the anti-NKG2A antibody is administered ata dose of 2-10 mg/kg, optionally 4-10 mg/kg, optionally 6-10 mg/kg,optionally 8-10 mg/kg, optionally 2-4 mg/kg, optionally 4-6 mg/kg,optionally 4-8 mg/kg, optionally 6-8 mg/kg body weight. Optionally, atleast 2, 3, 4, 5, 6, 7 or 8 doses of the anti-NKG2A antibody areadministered. In one embodiment, the administration cycle is between 2weeks and 8 weeks. In one embodiment, the administration cycle is 8weeks. In one embodiment, the administration cycle is 8 weeks andcomprises administering one dose of the anti-NKG2A antibody every twoweeks (i.e. a total of four doses).

In one aspect of any of the embodiments herein, the anti-NKG2A antibodyis administered once about every two weeks.

Suitable treatment protocols for treating a human include, for example,administering to the patient an effective amount of an anti-NKG2Aantibody, wherein the antibody is administered 2 times per month and theamount effective to maintain a continuous blood concentration ofanti-NKG2A antibody of at least 10 μg/ml between at least two successiveadministrations of the anti-NKG2A antibody is between 2-10 mg/kg,optionally 2-6 mg/kg, optionally 2-8 mg/kg, optionally 2-4 mg/kg,optionally 2-3 mg/kg, or optionally about 2, 3 or 4 mg/kg body weight.These doses can optionally be selected so as to provide for a continuedblood concentration of anti-NKG2A antibody of at least 10 μg/mlthroughout the treatment cycle. Achieving blood concentration ofanti-NKG2A antibody of 10 μg/ml corresponds to the EC₁₀₀ for an antibodysuch as humanized Z270.

Further suitable treatment protocols for treating a human include, forexample, administering to the patient an effective amount of ananti-NKG2A antibody, wherein the antibody is administered two times permonth and the amount effective to maintain a continuous bloodconcentration of anti-NKG2A antibody of at least 40 μg/ml between atleast two successive administrations of the anti-NKG2A antibody isbetween 2-10 mg/kg, optionally 2-8 mg/kg, optionally 2-6 mg/kg,optionally 2-4 mg/kg, optionally 2-3 mg/kg, or optionally about 2, 3 or4 mg/kg body weight. These doses can optionally be administered so as toprovide for continued blood concentration of anti-NKG2A antibody of atleast 40 μg/ml throughout the treatment cycle. Achieving bloodconcentration of anti-NKG2A antibody of 40 μg/ml is expected to providea tissue (e.g., extravascular tissue, tumor environment of a solidtumor) concentration of about 4 μg/ml, in turn corresponding to the EC₅₀for NKG2A+ NK cell response for an antibody such as humanized Z270.

Further advantageous suitable treatment protocols for treating a humaninclude, for example, administering to the patient an effective amountof an anti-NKG2A antibody, wherein the antibody is administered 2 timesper month and the amount effective to maintain a continuous bloodconcentration of anti-NKG2A antibody of at least 100 μg/ml between atleast two successive administrations of the anti-NKG2A antibody isbetween 4-10 mg/kg, optionally 4-6 mg/kg, optionally 4-8 mg/kg,optionally about 4 mg/kg, optionally about 6 mg/kg, optionally about 8mg/kg, or optionally about 10 mg/kg body weight. These doses canoptionally be administered so as to provide for continued bloodconcentration of anti-NKG2A antibody of at least 100 μg/ml throughoutthe treatment cycle. Achieving blood concentration of anti-NKG2Aantibody of 100 μg/ml is expected to provide a tissue (e.g.,extravascular, tumor environment) concentration of about 10 μg/ml, inturn corresponding to the EC₁₀₀ for an antibody such as humanized Z270.

Further advantageous suitable treatment protocols for treating a humanhaving cancer include regimens that employ a loading period with ahigher dose, followed by a maintenance period. For example, a loadingperiod may comprise administering to the patient an effective amount ofan anti-NKG2A antibody, wherein the antibody is administered one or moretimes in an amount effective to maintain a continuous bloodconcentration of anti-NKG2A antibody of at least 100 μg/ml until thefirst administration of anti-NKG2A antibody in the maintenance regimen.For example, when administered once, a loading dose of 10 mg/kg ofanti-NKG2A antibody can be administered, wherein the firstadministration of anti-NKG2A antibody within the maintenance regimenoccurs about two weeks (or less) after the loading dose. The maintenanceregimen can then employ a lower dose and/or lower frequency ofadministration in order to maintain a continuous blood concentration ofanti-NKG2A antibody of at least 100 μg/ml between successiveadministrations within the maintenance regimen. For example, amaintenance regimen can comprise administering anti-NKG2A antibody everytwo weeks at a dose of between 2-10 mg/kg, optionally 4-10 mg/kg,optionally 2-4 mg/kg, optionally 4-6 mg/kg, optionally 4-8 mg/kg,optionally about 4 mg/kg, optionally about 6 mg/kg, optionally about 8mg/kg body weight.

In one aspect, anti-NKG2A antibody is dosed in an amount so as to obtaina concentration in blood (serum) and/or tissue (e.g. tumor tissue) thatcorresponds to at least the EC₅₀ for NKG2A+ NK cell response, optionallyat about or at least about, the EC₁₀₀, for a period of at least about 1week, at least about 2 weeks, without a significant “de-saturation” ofNKG2A on cells in circulation between at least two successiveadministrations of the anti-NKG2A antibody. In another aspect, antibodyis dosed in an amount so as to obtain a concentration in blood (serum)and/or tissue (e.g. tumor tissue) that corresponds to at least the EC₅₀for NKG2A+ NK cell response, optionally at about or at least about, theEC₁₀₀, for a period of at least about 1 week, at least about 2 weeks,and that permits a significant “de-saturation” of NKG2A on cells incirculation between two (or between each) successive administrations ofthe anti-NKG2A antibody. For example, the anti-NKG2A antibody may beadministered in an amount and at a frequency may result in at least 50%de-saturation of NKG2A on cells in circulation during the treatmentperiod, for example between two successive administrations of theanti-NKG2A antibody. In one example, the amount that that permits asignificant “de-saturation” of NKG2A on cells in circulation is anamount that provides a blood concentration of less than the EC₅₀ forNKG2A+ NK cell response, optionally less than the EC₂₀ for NKG2A+ NKcell response.

Advantageous suitable treatment protocols for treating a human include,for example, administering to the patient an effective amount of ananti-NKG2A antibody, wherein the antibody is administered in an amounteffective to achieve a blood concentration of anti-NKG2A antibody of atleast 10 μg/ml, 40 μg/ml or 100 μg/ml for a period of at least about 1week, or at least about 2 weeks, and that permits (e.g. is followed by)a significant “de-saturation” of NKG2A on cells in circulation betweentwo (or between each) successive administrations of the anti-NKG2Aantibody.

For example, wherein the antibody is administered no more than once permonth (or no more than once about every two months) and the amounteffective to achieve a blood concentration of anti-NKG2A antibody of atleast 10 μg/ml, 40 μg/ml or 100 μg/ml for a period of at least about 1week, or at least about 2 weeks is between 2-10 mg/kg, optionally 2-8mg/kg, optionally 2-6 mg/kg, optionally 2-4 mg/kg, optionally 2-3 mg/kg,or optionally about 2, 3 or 4 mg/kg body weight.

Saturation (and de-saturation) of NKG2A on cells in circulation can beassessed by obtaining a peripheral blood sample and using standardmethods for assessing receptor occupancy.

In another aspect, provided is a method of reducing the risk of cancerprogression, reducing the risk of further cancer progression in a cellpopulation that has undergone initiation, and/or providing a therapeuticregimen for reducing cancer progression in a human patient, whichcomprises administering to the patient one or more first treatments(e.g. induction therapy, such as a chemotherapeutic agent) in an amountand regimen sufficient to achieve a response (partial or completeresponse), and then administering to the patient an amount of ananti-NKG2A antibody in a dosage and frequency according to thedisclosure.

In a further aspect, provided is a method of promoting remission of acancer in an individual, such as a human patient, comprisingadministering a composition comprising an anti-NKG2A antibody, to theindividual, in a dosage and frequency according to the disclosure, so asto promote cancer remission in the individual. In a further aspect,provided is a method of preventing recurrence of a cancer in anindividual, such as a human patient, whose cancer is in remissionfollowing a preceding anti-cancer treatment, comprising administering tothe individual a composition comprising an anti-NKG2A antibody, in adosage and frequency according to the disclosure, so as to promotecancer remission in the individual.

In an even further aspect, provided is a method for reducing the risk ofdeveloping a cancer, reducing the time to onset of a cancerouscondition, and/or reducing the severity of a cancer diagnosed in theearly stages, comprising administering to an individual aprophylactically effective amount of an anti-NKG2A antibody in a dosageand frequency according to the disclosure, so as to achieve the desiredphysiological effect(s).

In a further aspect, provided is a method of increasing the likelihoodof survival over a relevant period in a human patient diagnosed withcancer. In another aspect, provided is a method for improving thequality of life of a cancer patient comprising administering to thepatient a composition in an amount effective to improve the quality oflife thereof. In a further aspect, methods described herein can beapplied to significantly reduce the number of cancer cells in avertebrate host, such that, for example, the total number of cancercells is reduced. In a related sense, provided is a method for killing(e.g. either directly or indirectly causing death of) cancer cells in avertebrate, such as a human cancer patient.

The anti-NKG2A antibody can be administered as monotherapy or inadjunctive or combined administration (co-administration) with a secondtherapeutic agent, e.g. an anti-EGFR antibody. The adjunctive orcombined administration includes simultaneous administration of thecompounds in the same or different dosage form, or separateadministration of the compounds (e.g., sequential administration). Thus,the anti-NKG2A and second therapeutic agent can be simultaneouslyadministered in a single formulation. Alternatively, the anti-NKG2A andsecond therapeutic agent can be formulated for separate administrationand are administered concurrently or sequentially. The secondtherapeutic agent will normally be administered in amounts and treatmentregimens typically used for that agent in a monotherapy for theparticular disease or condition being treated.

EXAMPLES Example 1—Biacore Analysis of Humanized Z270

Humanized Z270 (humZ270) is described in U.S. Pat. No. 8,206,709 (NovoNordisk), the disclosure of which is incorporated herein by reference.As described in U.S. Pat. No. 8,206,709, HumZ270 VKI_O2/JK4 light chainand various heavy chain acceptor frameworks were produced as human IgG4antibodies. Heavy chain frameworks included “VH1” based on VH1_18/JH6;“VH5” based on VH5_a; “VH6” based on VH5_51; “VH7” based on VH1_f; and“VH8” based on VH1_46, all with a JH6 J-segment. The antigen-bindingproperties were analyzed on a Biacore T100 (Biacore AB, Uppsala,Sweden). The antigen was in the form of a single-chain NKG2A-CD94-mFcconstruct was covalently immobilized on the sensor CM5 chip (Biacore AB,Uppsala, Sweden) via amine groups using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS). The immobilization level was targeted at 300RU. Z270 antibody variants were diluted to a concentration series(0.157, 0.313, 0.625, 1.25, 2.5 nM) in the running buffer HBS-EP (10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Tween-20). All thesamples were then injected over the immobilized antigen for 2 min at theflow rate of 40 ul/min. Subsequently, the running buffer was injectedfor 3 min at 40 ul/min for antibody dissociation analysis. After eachrun, the regeneration buffer (10 mM NaOH, 500 mM NaCl) was injected (30seconds, 10 ul/min) to completely strip the remaining antibodies off theantigen. Data were evaluated with Biacore T100 evaluation software.

The affinity of humanized Z270 VH1 was determined as 67 pM. Affinitiesof the VH5, VH6, VH7 and VH8 were comparable.

TABLE humZ270 ka (1/Ms) Kd (1/s) KD (M) Chi² (RU²) 7.492E+6 4.982E−46.650E−11 0.044

Example 2—humZ270 is a Competitive CD94/NKG2A Antagonist

As described in U.S. Pat. No. 8,206,709, to test whether humZ270prevents ligand (i.e. HLA-E) binding to CD94/NKG2A, humZ270 was testedfor the ability to prevent the binding of HLA-E tetramers to CD94/NKG2Aover-expressing Ba/F3 cells (Ba/F3-CD94/NKG2A). humZ270VL1/VH1 havingthe respective heavy and light chains of SEQ ID NOS: 3 and 7 was used inthis Example, and unless indicated to the contrary, this antibody wasalso used as humZ270 for all other Examples below. For this,Ba/F3-CD94/NKG2A were incubated with 1) various concentrations ofhumZ270 or 2) first incubated with a saturating concentration of HLA-Etetramers (4.7 μg/ml) and then incubated with various concentrations ofhumZ270. All incubations were performed in tissue-culture mediumcontaining 2% FCS, on ice. Subsequently, cells were incubated withAPC-conjugated secondary antibodies specific for mouse Ab's, andanalyzed by flowcytometry using a BD Biosciences FACSarray.

humZ270 efficiently binds Ba/F3-CD94/NKG2A cells in a concentrationdependent fashion (diamonds). However, when cells were pre-incubatedwith HLA-E tetramers, humZ270 was prevented from binding toBa/F3-CD94/NKG2A cells. Thus HumZ270 and HLA-E bind overlapping epitopeson CD94/NKG2A. Therefore, the CD94/NKG2A-inhibitory effect of humZ270 inNK-cytotoxicity assays is likely a consequence of preventing the abilityof HLA-E inducing negative signals to cytotoxic lymphocytes viaCD94/NKG2A. As such, humZ270 can be considered a competitive CD94/NKG2Aantagonist.

Example 3—Distribution of NK and T Cell Subsets in C57/Bl6 Mice BearingRma-Rae1 Tumor Based on NKG2A Expression

To further investigate the expression of NKG2A in tumor settings,distribution of NKG2A was studied on NK and T cell subsets in mice.Lymphocytes were taken from spleen, from tumor draining lymph nodes, aswell as from within solid tumors.

C57/BL6 mice were engrafted (sc) with RMA-Rae clone 6 (2 million cells).These tumor cells express CD94/NKG2A ligand, Qa-1. Mice were sacrificedat day 12 with a mean tumor volume: 723 mm³, SD: 161 mm³, n=4. Followingcell suspension preparation from spleen, LN and tumor, cells werestained as follows: CD3e PerCP Cy5.5, NKP46 Alexa 647, NKG2A/C/E FITC,CD8 Pacific Blue.

Results, shown in Tables 1-3, revealed NKG2A-expressing NK and CD8+ Tlymphocytes are found in significant percentages within the tumorenvironment with NKG2A+ CD8+ T cells being present in high percentagesin the tumor environment but not in the spleen or tumor draining lymphnodes. In the NK cell subset, cells in both the draining lymph nodes andspleen were about half NKG2A-positive and half NKG2A-negative. In the Tcell subset most cells were NKG2A-negative (only 1.1% in lymph nodes and4.7% in spleen are NKG2A⁺). In the CD8 T cell subset, most cells wereagain NKG2A-negative (only 1.6% in lymph nodes and 3.9% in spleen areNKG2A⁺). However, while almost no CD8 T cells outside the tumor hadNKG2A expression, tumor infiltrating CD8 T cell subset had a mean of26.3% NKG2A+ positive cells. Among the CD8⁻ T cell subset, there waslittle difference in NKG2A expression observed between TILs and spleenor lymph node cells, as only 5.1% of CD8⁻ T cells in the tumor expressedNKG2A.

TABLE 1 Spleen % NK % T % T CD8+ NKG2A+ NKG2A+ NKG2A+ Mice1 Spleen41.863 4.54 4.95 Mice2 Spleen 46.198 6.25 3.36 Mice4 Spleen 44.49 3.373.45 Mean 44.2 4.7 3.9 SD 2.2 1.45 0.89

TABLE 2 Tumor Draining Lymph Nodes % NK % T % T CD8+ NKG2A+ NKG2A+NKG2A+ Mice1 LN 45.95 0.5 2.1 Mice3 LN 55.10 0.7 3.1 Mice4 LN 49.11 0.60.8 Mean 50.05 1.1 1.6 SD 4.65 1.1 1.3

TABLE 3 Tumor Infiltrating Lymphocytes % NK % T % T CD8+ NKG2A+ NKG2A+NKG2A+ Mice1 TIL 52.2 2.89 26.8 Mice2 TIL 44.5 8.22 26.05 Mice3 TIL 529.3 34.31 Mice4 TIL 55 0 17.9 Mean 50.9 5.1 26.3 SD 4.5 4.4 6.7

Example 4—In Vitro Receptor Saturation: Binding of Humanized Anti-NKG2AAntibody PH2201 to NKG2A+ NK Cells in Whole Blood

Receptor occupancy of anti-NKG2A was assessed in vitro in human blood,providing a prediction of the pharmacokinetics of anti-NKG2A in humanpatients. This study was aimed to estimate ex vivo the cellular affinityin whole blood of HumZ270 as well as the absolute number of availableNKG2A receptors per μl of whole blood and also per cell in humans. Theaffinity and total number of receptors may impact both pharmacokinetics(PK) and pharmacodynamics (PD) of the mAb in humans. These data will beused as input for the PK/PD model which will be used for designing thefirst human dose trial. Human whole blood was collected from each donorand processed immediately. A titration of humZ270 was done in wholeblood from 8 volunteers (mAb incubated for 30 minutes at roomtemperature (RT). Fifteen mAb concentrations were tested to cover thefull range of binding between 0 and 100% of maximal binding, going from90 μg/ml to 0.000019 μg/ml (1/3 serial dilution, 15 points and 0).Samples were processed to remove red blood cells and fix the cells andacquired on a cytometer. Calibrating beads were used to transform MFIresults into MESF (Molecule of Equivalent Soluble Fluorochrome). Gatingwas done on CD45+ lymphocytes expressing NKG2A.

Three tubes were dedicated to absolute counting of lymphocyte subsetsusing flow-count beads added into the blood. First, a titration was doneusing humZ270, in which bound humZ270 was be detected using a PE-coupledanti-hIgG4 secondary antibody. A titration was also performed withPE-coupled humZ270 and compared to titration done with bound humZ270 inorder to examine the effect of the coupling to PE on the pharmacologicalproperties of humZ270 and for evaluating the total number of receptorsfor a saturating concentration of humZ270.

Analysis focused on NKG2A+ cell subsets, i.e. NK cells and T cells.These subsets of lymphocytes do not all express NKG2A and therefore aNKG2A-subset for each population of interest were studied and comparedto the NKG2A+ subset. The cell populations are defined as follows:

-   -   Lymphocytes: defined on a CD45/SSC plot, according to their        granularity and size and CD45 expression    -   Human NK lymphocytes: CD3-CD56+ cells among lymphocytes    -   CD8 T cells: CD3+ CD8+ cells among lymphocytes    -   NKG2A+ lymphocytes: NKG2A+ cells among lymphocytes.

The total Mean Fluorescence Intensity (MFI) was recorded for eachpopulation. Calibrating beads was used to express Fluorescence in PEchannel as MESF. The main populations of interest in this study areNKG2A+ lymphocytes mainly including NK cell.

Results showed that human anti-NKG2A mAb (humZ270) binds to two subsetsof peripheral blood lymphocytes: NK and CD8+ T cells.

Results are summarized in FIG. 1. HumZ270, also referred to as IPH2201,is shown at different concentrations (ng/ml) listed on the x-axis andMESF signal for receptor binding is shown on the y-axis. The antibodyhad a binding affinity (EC50) of about 4 ng/mL for NKG2A+ cells (K_(D)˜4ng/mL). This K_(D) is consistent with K_(D) values observed in otherassays, notably affinity for binding to PBMC and affinity forrecombinant NKG2A in Biacore assays. The K_(D) for full receptoroccupancy (the EC100) was about 100 ng/ml.

Example 5—Receptor Saturation of IPH2201 in a Human Phase I ClinicalTrial in Rheumatoid Arthritis

HumZ270, a human IgG4 antibody was produced with a heavy chain having aserine to proline mutation in residue 241 of the heavy chain (S241Pmutation), corresponding to position 228 according to the EU-index, inorder to maintain high affinity bivalent binding in vivo. The safetyprofile of humZ270 (IPH2201) was explored in a double-blind,placebo-controlled dose-escalation phase I trial in 92 patients withstable and controlled rheumatoid arthritis. In this three-armed phase Itrial, Z270 or placebo was administered as single-dose i.v. up to 10mg/kg, or single-dose s.c. or multiple-dose s.c. (four administrationsgiven with 2-week intervals) up to 4 mg/kg. The dosages tested as singledose by i.v. were: 0.0002, 0.001 mg/kg, 0.005 mg/kg, 0.025 mg/kg, 0.1mg/kg, 0.4 mg/kg, 1.1 mg/kg, 3.5 mg/kg and 10 mg/kg. All patients werefollowed for a minimum of 12 weeks. The safety profile was veryfavorable. The MTD was not reached. There were no SUSAR, no drug-relatedserious adverse events, no infusion related reaction and no immunerelated adverse event. Nasopharyngitis and headache were the two mostfrequently reported adverse events.

Receptor saturation was assessed using a conventional sandwich ELISAformat with a mouse Fc human NKG2A fusion protein used to capture thehumZ270 antibody. After incubation with serum samples containing humZ270antibody, bound antibody (the analyte in this study) is detected using abiotinylated mouse anti human IgG4. HRP labelled avidin is added to tagthe solid phase bound biotinylated anti-human IgG4 and a colorimetricHRP substrate (TMB) is used for end point detection. The optimal doseand dosing frequency for the Anti-NKG2A (IPH2201) dose levels to be usedin the clinical trials that would provide substantially full receptorsaturation was predicted using a PK/PD model developed usingpharmacokinetic software package (WinNonLin 6.3.0.395, PharsightCorporation), model based on clinical data from IPH2201 phase I in RApatients. The dosing frequency in clinical therapy using IPH2201 dependson the steady state plasma concentration needed for saturation as wellas the clearance and volume of distribution of IPH2201.

Preliminary PK data and NCA analysis for IPH2201 phase I clinical trial,i.v. injection, enabled us to estimate the PK parameters for the PKmodel. Nominal times were used. A standard 2-compartment model wasselected, with 1^(st) order elimination and a non Linear Target MediatedDrug Disposition modelled by Michaelis Menten kinetic. A dose-dependentclearance was observed in NCA analysis and applied to this model, withclearance decreasing with increasing doses under 0.4 mg/kg, thenremaining constant for higher doses. This preliminary PK model isconsistent with known PK characteristics for IgGs for which dosedependency reflects target receptor (at low doses) and FcRn receptor(high doses) saturations (Brambell).

A population PD modelling was performed based on preliminary NKG2Aoccupancy data available from the ongoing phase I clinical trial.

A sigmoid Emax model was used to fit the plasma concentration-effectrelationship. The model was characterized by an EC₅₀, defined as theserum drug concentration to reach 50% of E_(max), i.e. 50% of NKG2Aoccupancy here.

However, when PD data were plotted against PK data, it was observed thatas time after dosing increased, maximum NKG2A occupancy (Emax) obtainedfor the highest IPH2201 concentrations decreased in a time dependentlinear manner, with no impact of the dose. Similarly, the EC50 seemed tolinearly increase with time. In order to model this observation in NKGA2occupancy profile, a decrease in Emax and an increase in EC50 with timewere included to describe the NKG2A occupancy data.

% NKG2Aoccupancy=(E_(max−)(E_(max)Fall×TSLD))×C^(γ)/((EC₅₀+EC₅₀inc×TSLD)^(γ)+C^(γ))

Where TSLD=Time Since Last Dose; E_(max)=100%; E_(max)F_(al)=Rate ofDecrease in Emax with time; EC₅₀inc=Rate of Increase in EC₅₀

Results of modelling are shown in FIG. 2, substantially complete (atleast 90%) receptor saturation of NKG2A+ cells can be achieved byadministering 0.03 mg/kg every two weeks. A dose of 0.1 mg/kg can beadministered every four weeks to maintain substantially completesaturation of NKG2A. If saturation in the extravascular tissue (e.g. asolid tumor) is desired, an approximately 10-fold higher dose isbelieved to be needed, translating to a dose of about 0.4 mg/kg everytwo weeks, 1 mg/kg every four weeks.

The results for saturation of NKG2A receptors from this human clinicaltrial was consistent with the in concentration of anti-NKG2A requiredfor full receptor occupancy (the EC100) determined in vitro (see Example4).

Example 6—CD107 Response to NKG2A Blockade Using IPH2201

Autologous in vitro experiments using effector and target cells wereperformed in order to assess the killing mediated by cytokine-activatedpurified NK cells (effector cells) in 12 human individuals. For threedonors, assessment of CD107 mobilization and occupancy was performed inparallel. The target cells were autologous SEB blasts. This killing isconsidered to be mediated by NK cells, and it can be specificallyincreased by an anti-NKG2A antibody as it is expected to counteract theinhibitory signal from HLA-E. A concentration-range response withantibody humZ270 was performed in a 4-hour cytotoxicity assay. It waschosen to use purified NK cells and a CD107 mobilization assay based onthe experimental results generated. First, the FACS-based CD107 assayenables the specifically study of the cytotoxic response of NK cellsexpressing NKG2A and to compare it to NK cells not expressing NKG2A. Analternative protocol such as chromium release assays would not haveallowed discriminating the effect depending on NKG2A expression which isan issue in donors with a low percentage of NKG2A+ cells.

Autologous SEB blasts were generated by incubating frozen PBMC with 200IU/ml recombinant human IL-2 (Proleukin) and 100 ng/ml SEB(Staphylococcal Enterotoxin B, Sigma) for 4 days in complete medium(RPMI, 10% FCS, Penicillin/Streptomycin). Then CD4 T cells werenegatively selected by using the “Stemsep negative selection human CD4 Tcell enrichment kit” (article number #14052A). Purity was assessed byFACS and HLA-E expression is measured with the PE-conjugated anti-HLA-Eclone 3D12 (E-Biosciences). T CD4 SEB blasts were considered as a goodmodel for generating autologous cells mimicking autoreactive T CD4+cells.

Purified NK cells were prepared from frozen PBMC: Human NK purificationwith Stem Sep system, and cultured O/N in complete medium supplementedwith 10 IU/ml recombinant IL-2. PBMC were prepared from collected blood.Blood was qualified for transfusion, donors were thus anonymous andsupposedly healthy. PBMC were prepared, aliquoted and stored in nitrogentanks prior to the study.

Calculations of Kd for receptor saturation and EC₅₀ for CD107mobilization were performed in GraphPad Prism software using anon-linear regression fit with four parameters. In order to estimate theaffinity constant Kd from the titration curves of anti-NKG2A (IPH2201)binding to purified NK cells, values for the 30 and 90 μg/ml wereexcluded because some artifacts were observed at these concentrations.

Results are summarized in FIG. 3A. Surprisingly, the concentrations ofanti-NKG2A required for efficacy (blockade) are 100-fold higher thanconcentrations providing full receptor saturation. The EC50concentration (the amount that provides a 50% of maximum) in the CD107mobilization assay was determined to be about 400 ng/ml, and the EC100was about 10,000 ng/ml (10 μg/ml). In contrast, as shown in Example 4,the EC50 concentration for receptor saturation is about 4 ng/ml and theEC100 is about 100 ng/ml, a concentration also corroborated by data fromthe Phase I clinical trial (see Example 5).

For the three donors with parallel assessment of CD107 mobilization andoccupancy, the individual EC₅₀ values for CD107 were approximately100-fold higher than the individual Kd for receptor saturation. The sameratio was observed when evaluating all data (median Kd for receptorsaturation 0.005 μg/ml, n=5, median EC50 for CD107 mobilization 0.40μg/ml, n=9). This suggests that donors generally have comparablereceptor affinities for IPH2201 and that the relationship betweenreceptor saturation and biological activity is similar.

Example 7—Effect of HLA-E Expression Levels on CD107 Response to NKG2ABlockade

The 100-fold difference between Kd obtained for receptor saturation andthe EC for CD107 mobilization could potentially be explained bymembrane-bound HLA-E on the target cells in the CD107 assay whichengages NKG2A on the NK cells with a high avidity, requiring moreIPH2201 to reach functional saturation of the receptor compared toreceptor saturation experiments in which there is no HLA-E involved.

To investigate this possibility, the EC observed for biological effectin autologous SEB blasts in Example 6 was compared to that of cellshaving different levels of expression of HLA-E. Human K562 cellstransfected with HLA-E were selected as high HLA-E expressing cells. TheFACS-based CD107 assay was performed using purified NK cells asdescribed in Example 6.

In each experiment, autologous CD4+ SEB blasts and a selected K562-HLA-Eclone were tested for HLA-E expression using the anti-HLA-E mAb, 3D12,coupled to PE. HLA-E expression on K562HLA-E transfectants was at least20 fold higher than the constitutive HLA-E expression observed onautologous SEB blasts. The purity of the SEB blasts was 96%±0.818 (SD,n=12, range 94.6%-97.1%). The expression of HLA-E on T CD4 SEB blastsand K562-HLA-E was similar between all donors.

Corroborating this observation of a 100-fold difference between Kdobtained for receptor saturation and the EC for CD107 mobilization, thelower EC observed for biological effect on the SEB blasts (median 0.40μg/ml) compared to the EC50 on K562-HLA-E transfectants (median 4.1μg/ml) can be explained by the at least a 20-fold difference in HLA-Eexpression at the cell surface. In this case, as HLA-E is expressed athigher densities on K562-HLA-E transfectants, higher amount of IPH2201are required to compete with HLA-E when compared to autologous SEBblasts.

Example 8—Pharmacokinetic Model Prediction for Repeated Injections ofIPH2201

Based on the EC for CD107 mobilization observed for autologous (SEB)cells in Example 7, and with the incorporation of the bloodconcentration results from the Phase I trial described in Example 5,modelling was performed to determine the optimal dosing frequency forthe Anti-NKG2A (IPH2201) dose levels to be used in the clinical trialsthat would provide efficacy.

The optimal dose and dosing frequency for the anti-NKG2A (IPH2201) doselevels to be used in the clinical trials was predicted using a PK/PDmodel developed using pharmacokinetic software package (WinNonLin6.3.0.395, Pharsight Corporation), model based on clinical data fromIPH2201 phase I in RA patients. The dosing frequency in clinical therapyusing IPH2201 depends on the steady state plasma concentration needed aswell as the clearance and volume of distribution of IPH2201.

Preliminary PK data and NCA analysis for IPH2201 phase I clinical trial,i.v. injection, enabled us to estimate the PK parameters for the PKmodel. Nominal times were used. A standard 2-compartment model wasselected, with 1^(st) order elimination and a non Linear Target MediatedDrug Disposition modelled by Michaelis Menten kinetic. A dose-dependentclearance was observed in NCA analysis and applied to this model, withclearance decreasing with increasing doses under 0.4 mg/kg, thenremaining constant for higher doses. This preliminary PK model isconsistent with known PK characteristics for IgGs for which dosedependency reflects target receptor (at low doses) and FcRn receptor(high doses) saturations (Brambell).

Based on preliminary PK and PD data from clinical phase I trial, thefollowing PK and PD parameters best fitted the observed data:

Dose Level CL (L/h) Parameter Value Unit  0.02 0.018 Ka  0.00426 h-1 0.04 0.015 F (Biodispo)  0.826  0.2 0.010 CL for 1 mg/kg  0.006 L/h 0.4 0.008 V1  2.8 L  2 0.006 V2  1.99 L  4 0.006 Q  0.0109 L/h 10 0.006Vnn  0.223 mg/wk Km 21.7 ng/ml

FIG. 3B shows different blood concentrations reached with differentdosages administered by i.v. every two weeks. Dose levels were 0.01,0.02, 0.03, 0.04, 0.2, 0.4 mg/kg, 2 mg/kg, 4 mg/kg and 10 mg/kg bodyweight. The lines in FIG. 3B correspond, from bottom to top, toincreasing doses of IPH2201, where the lowest dose corresponds to thelowest line at the bottom of the chart to the highest dose at the top ofthe chart. The dose of 0.01 mg/kg provided an initial (peak) bloodconcentration of about 0.2 μg/ml. The dose of 0.04 mg/kg provided aninitial (peak) blood concentration of about 1 μg/ml, i.e. above the EC₅₀for efficacy of 400 ng/ml, but after two weeks was below the EC₅₀. Thedose of 0.4 mg/kg provided an initial (peak) blood concentration ofabout 10 μg/ml, i.e. at about the EC₁₀₀ for efficacy in circulation, anda continued blood concentration above 3 μg/ml up to the two week timepoint. The dose of 4 mg/kg provided an initial (peak) bloodconcentration of about 100 μg/ml, i.e. at about the EC₁₀₀ for efficacyin tissues, and a continued blood concentration above 30 μg/ml up to thetwo week time point. However, when the dose of 4 mg/kg is administeredin repeat dosing, the fourth dose provides continued blood concentrationof approximately 100 μg/ml. The dose of 10 mg/kg provided a continuedblood concentration approximately 100 μg/ml.

FIG. 3C shows different blood concentrations reached with differentdosages administered by i.v. every two weeks, when a loading dose andmaintenance dose are used. In a two-week dosing frequency, a loadingdose of 10 mg/kg body weight followed by a maintenance dose of 4 mg/kgbody weight (the upper line in FIG. 3C) provides provided an initial andcontinued blood concentration approximately 100 μg/ml. For purposes ofcomparison, a constant dose of 4 mg/kg is shown as the lower line inFIG. 3C, which provides for a continued (or minimal) blood concentrationof at least 30 μg/ml between the initial and subsequent injection at twoweeks, a continued (or minimal or remaining) blood concentration of atleast 50 μg/ml at two weeks after the second injection, a continued (orminimal or remaining) blood concentration of at least 60-70 μg/ml at twoweeks after the third injection.

Example 9—Effect of a Dose-Response of Anti-NKG2A on NK Cells Activation

Immunotherapeutic approaches for HNSCC are particularly complicated bythe profound immune suppression that is induced by HNSCC, whichpotentially decreases the effectiveness of immune stimulatory efforts(see, e.g., Duray et al. (2010) Clin. Dev. Immunol. 2010: 1-15). Thegoal of this experiment was to explore whether an anti-NKG2A antibodythat targets NKG2A is able to eliminate HNSCC cells.

Effect of a dose-response of anti-NKG2A on NK cells activation wasdetermined by analysis of CD107 and CD137 expression. CD107 mobilizationat 4 hours is a marker of the release of lytic granules by NK cells(Alter et al., (2004) J Immunol Methods 294(1-2): 15-22). Increase inCD137 expression at 24 hours is correlated with the activation ofseveral lymphocytes including NK cells (Kohrt et al. (2011) Blood117(8):2423-2432). Analysis of CD107 and CD137 expression was performedon NK cells expressing or not expressing NKG2A (NKG2A+ NK cells orNKG2A-NK cells respectively). Since the antibody is targeting NKG2A, itseffect is expected to be seen only on NKG2A+ NK cells, and thus NKG2A-NKcells can be regarded as an internal control in the experiments.

The effector cells used were freshly isolated PBMC from healthyvolunteers and target cells were HNSCC cell lines or clones of K562 cellline transfected with HLA-E. Cells were numerated and passed every twodays in complete medium. Viability was measured and had to be over 90%.They were kept in culture up to 12 passages. The day before of theexperiment, cells were counted and adjusted to 100,000 cells/wells.Viability was measured and had to be over 90%.

The K562 cell lines were K562 clone E6 (HLA-E positive, CD32low) andK562 clone F7 (HLA-E negative/low, CD32low). Human head and neck cancercell lines were screened by flow cytometry for HLA-E expression (seeTable C, below). Three cell lines were selected for functional tests:FaDu (ATCC # HTB-43), H-N (DSMZ # ACC 417) and CAL-27 (DSMZ # ACC 446).

TABLE C HLA-E expression Mean HLA-E/ # Cell line IC MFI ratio exp. Celltype Source H-N 2.8 n = 2 Oral squamous cell carcinoma DSMZ, GermanyDetroit 562 2.7 n = 1 Pharynx carcinoma ATCC, USA (metastatic site:pleural effusion) SCC-9 3.7 n = 1 Tongue squamous carcinoma ATCC, USAA-253 3.5 n = 1 Submaxillary salivary gland; epidermoid ATCC, USAcarnoma FaDu 5.2 n = 2 Pharynx squamous cell carcinoma ATCC, USA BICR61.8 n = 1 Hypopharynx squamous cell carcinoma Public Health England, UKBICR16 2.4 n = 1 Tongue squamous carcinoma Public Health England, UKCAL-27 4.0 n = 2 Tongue squamous carcinoma DSMZ, Germany BICR10 2.3 n =1 Buccal mucosa squamous carcinoma Public Health England, UK

The effector cells used were freshly isolated PBMC from healthyvolunteers. Target cells were the HNSCC cell lines (FaDu, H-N andCAL-27), and clones of K562 cell line transfected with HLA-E (CloneE6=HLA-E⁺, clone F7=HLA-E) as an E:T ratio 2.5/1. Read out was CD107 at4 hours vs. CD137 at 24 hours. 7 donors (FaDu and H-N) and 2 donors(CAL-27) were tested. Anti-NKG2A antibody humZ270 VH1 whose heavy chainamino acid sequence is shown in SEQ ID NO: 3 and whose light chain aminoacid sequence is shown in SEQ ID NO: 7 was used at a final concentrationof 10 μg/mL.

FIGS. 4A and 4B show CD107 (Top) and CD137 (Bottom) FACS read-outs onNKG2A-NK (left) or NKG2A+ NK cells (right) in presence of controls orindicated target cell lines and in presence or not of anti-NKG2A at aconcentration of 10 μg/mL. The cell lines are ordered from left to rightaccording to level of HLA-E surface expression. Each dot represents PBMCfrom a healthy volunteer. FIG. 4B shows HNSCC cell lines anddemonstrates anti-NKG2A can restore lysis of HNSCC with endogenous HLA-Eexpression or of K562 transfected with HLA-E. This effect is only seenon NKG2A positive NK cells and is dependent on the level of expressionof HLA-E. Indeed, anti-NKG2A effect is seen on cell lines with medium tohigh HLA-E level of expression.

Anti-NKG2A can induce the NK-mediated lysis of HLA-E expressing celllines by blocking the interaction of the inhibitory receptor NKG2A withHLA-E. This effect is observed on K562 cell line transfected with HLA-E,but more importantly on HNSCC cell lines with endogenous HLA-Eexpression such as FaDu and CAL-27 cell lines. The extent of anti-NKG2Aeffect depends on the level of HLA-E expression at the cell surface ofthe target cells.

Example 10—Combined Effect of an Optimal Dose of Anti-NKG2A withSub-Optimal Doses of Cetuximab

Epidermal growth factor receptor (EGFR), (also ErbB-1; HER1 in humans),is a ubiquitously expressed transmembrane glycoprotein in the ErbB/HERfamily of receptor tyrosine kinase. High expression of EGFR occurs inmost epithelial malignancies including HNSCC and is associated with apoor prognosis. Its activation through natural ligands leads to theinitiation of intracellular signaling pathways that regulate theactivation of cell proliferation, invasion, angiogenesis and metastasisdriving tumor growth.

The anti-EGFR monoclonal antibody cetuximab is thought to act throughblocking oncogenic signaling of the EGF receptor pathway and by inducingFcγ receptor-mediated antibody dependent cellular cytotoxicity (ADCC).In HNSCC however, ADCC may be affected by the profound immunesuppression that is induced. At the same time, blocking oncogenicsignaling of the EGF receptor pathway results in posttranscriptionalregulation in tumor cells of major histocompatibility complex (MHC)class I-related antigens of the MICA/B and ULBP protein families whichare recognized by the activating receptor NKG2D on NK cells and subsetsof T cells. In particular, the expression by tumor cells of thesestress-related antigens which are the natural ligands of NKG2D isdecreased by clinical EGFR inhibitors, thus potentially decreasing thetumor cells' visibility to NK and T cells (Vantourout et al., Sci.Transl. Med. 6: 231ra49 (2014).

This experiment was designed to explore the effect of an EGFR inhibitingantibody on the ability of anti-NKG2A antibodies to activate NK cells inHNSCC. Effect of a dose-response of cetuximab on NK cells activation wasdetermined by analysis of CD107 and CD137 expression, using as effectorcells PBMC freshly isolated from healthy volunteers, and as target cellsHNSCC cell lines (FaDu, and H-N), at an E:T ratio of 2.5/1. The read outwas CD107 at 4 hours vs. CD137 at 24 hours, using 3 donors (CD107 readout in 4h) and four donors (CD137 read out in 24h). Cetuximab was testedat a dose response, 1/10 serial dilution starting at 10 μg/mL. For onehealthy volunteer, NK cells were subdivided in NKG2A+ and NKG2A-subsets.Suboptimal doses of cetuximab were chosen for further testing to explorethe effect of an EGFR inhibiting antibody on the ability of anti-NKG2Aantibodies to activate NK cells in HNSCC. The 0.001 μg/mL (1 ng/mL) doseis the starting point of the cetuximab effect observed both with CD107and CD137 readouts. The 0.01 μg/mL (10 ng/mL) dose is approximately atthe EC50 of the cetuximab effect.

The combined effect of anti-NKG2A and a sub-optimal dose of EGFRinhibitor was assessed by analysis of CD107 and CD137 expression, usingas effector cells freshly isolated PBMC from healthy volunteers, and astarget cells HNSCC cell lines (FaDu, H-N and CAL-27), clones of K562cell line transfected with HLA-E (Clone E6=HLA-E⁺, clone F7=HLA-E), atan E:T ratio of 2.5/1. The read out was CD107 at 4 hours vs. CD137 at 24hours, using 7 donors (FaDu and H-N)) and two donors (CAL-27).

Anti-NKG2A antibody whose heavy chain amino acid sequence is shown inSEQ ID NO: 3 and whose light chain amino acid sequence is shown in SEQID NO: 7 was used at a final concentration of 10 μg/mL corresponding tofull functional activity, and EGFR inhibitor cetuximab was used at twosuboptimal doses of 0.001 μg/mL or 0.01 μg/mL.

Full activity concentrations of Anti-NKG2A enhanced ADCC to HNSCC celllines induced by suboptimal doses of cetuximab. No cetuximab-dependentADCC was observed on K562 transfected cell lines as they do not expressEGF-R. Anti-NKG2A effect is only seen on NKG2A positive NK cells, and isdependent on the level of expression of HLA-E. Indeed, anti-NKG2A effectis seen on cell lines with medium to high HLA-E level of expression(FaDu, CAL-27 and K562-HLA-E clone E6). FIG. 5 is a representativeexample, shown for FaDu cells. It can be seen in FIG. 5 that fullactivity doses of anti-NKG2A enhanced ADCC to HNSCC cell lines inducedby suboptimal doses of cetuximab (ctx).

Example 11—Combined Effect of Increasing Doses of Anti-NKG2A withIncreasing Doses of Cetuximab

The effect of the combination of increasing doses of EGFR inhibitingantibody an increasing doses of anti-NKG2A antibodies was evaluated forthe ability to activate NK cells toward HNSCC target cells. Experimentssought to evaluate whether anti-NKG2A therapy can still enhance ADCCwhen cetuximab is used at a saturating dose, and whether the anti-NKG2Aeffect is dose-dependent.

Briefly, effector cells used were freshly isolated PBMC from healthyvolunteers, and target cells were HNSCC cell lines (FaDu, H-N andCAL-27), and clones of K562 cell line transfected with HLA-E (CloneE6=HLA-E⁺, clone F7=HLA-E), and an E:T ratio of 2.5/1. The read out wasCD107 at 4 hours vs. CD137 at 24 hours, using 2 donors. Anti-NKG2Aantibody whose heavy chain amino acid sequence is shown in SEQ ID NO: 3and whose light chain amino acid sequence is shown in SEQ ID NO: 7 wasused at two suboptimal doses of 0.1 and 1 μg/mL, and at a saturatingdose of 10 μg/mL. Cetuximab was used at two suboptimal doses of 0.001μg/mL and 0.01 μg/mL (˜EC50) and at a saturating dose of 0.1 μg/mL inthese experimental settings.

Results are shown in FIGS. 6A and 6B. FIG. 6A shows CD107 read out oncontrols with no target and with K562-HLA-E transfectants. Each healthyvolunteer is represented by a different symbol: squares or circles.Crossed open symbols correspond to condition where anti-NKG2A wasreplaced by 10 μg/mL hIgG4 isotypic control co-incubated with 0.1 μg/mLcetuximab. It can be seen that the effect of anti-NKG2A antibody isdose-dependent, depends on HLA-E expression level, and is still observedat a saturating dose of cetuximab of 0.1 μg/mL in the clone E6 thatexpresses HLA-E at high levels. In clone F7 (low HLA-E expressionlevels), the effect of anti-NKG2A was limited and high doses (e.g. fullactivity doses) of anti-NKG2A did not further augment the effect.

FIG. 6B shows CD107 read out on HNSCC cell lines. Each healthy volunteeris represented by a different symbol: squares or circles. Crossed opensymbols correspond to condition where anti-NKG2A was replaced by 10μg/mL hIgG4 isotypic control co-incubated with 0.1 μg/mL cetuximab. Itcan be seen that the effect of anti-NKG2A antibody is dose-dependent,depends on HLA-E expression level, and is still observed at a saturatingdose of cetuximab of 0.1 μg/mL in the FaDu or CAL-27 that expresses athigher levels/stain strongly for HLA-E.

The effect of anti-NKG2A antibody is dose-dependent, depends on HLA-Eexpression level, and is still observed at a saturating dose ofcetuximab of 0.1 μg/mL.

Example 12—a Human Clinical Trial for Treatment of Cancer with RepeatedInjections of Humanized Z270 as Single Agent

The primary objective of the trial is to evaluate the antitumor activityof pre-operative IPH2201 (humanized Z270 comprising an S241P mutation)in patients with operable squamous cell carcinoma of the oral cavity.The secondary objectives are to assess the safety of IPH2201, thepharmacokinetics, the immunogenicity and the pharmacodynamics includingintra-tumoral biomarkers.

Trial Design:

The trial is a single-center, open label single-arm phase Ib-II studyincluding a run-in part. Previously untreated patients with measurable,clinical intermediate or high risk, stage III or IVa squamous cellcarcinoma of the oral cavity will be treated with single agent IPH2201i.v. every 2 weeks (q2w) for 4 administrations, by intravenous (i.v.)route over 1 hour. The first 6 patients will receive IPH2201 at a doseof 4 mg/kg q2w×4. A minimum interval of one week will be observedbetween the first administrations of IPH2201 to the 3 first patientstreated at 4 mg/kg. The subsequent patients will be treated at a dose of10 mg/kg q2w×4, the escalation of the dose being allowed by the safetycommittee after a minimal follow-up of 4 weeks following the firstadministration in the last patient treated at 4 mg/kg. Standardloco-regional treatment with surgery followed by adjuvant therapy(radiotherapy (RT) or radiochemotherapy (RCT)) according tohistopathologic risk factors will be initiated after the lastadministration of IPH2201. In case of tumor progression loco-regionaltreatment will be initiated immediately.

Antitumor activity will be assessed clinically and radiologically beforethe third administration of IPH2201, and 2 weeks after the lastadministration of IPH2201, before surgery. Tumor measurements will beobtained on target lesions according to RECIST 1.1 criteria. The sameimaging techniques will be used for efficacy assessment at baseline andduring the preoperative period, for the assessment of the primary endpoint and/or after surgery, for the monitoring of potential relapses. Anassessment by appropriate imaging techniques, at the investigator'sdiscretion (computed tomography (CT) scans and/or Magnetic ResonanceImaging (MRI)) will be performed in all the patients, as well asphotographs of the accessible tumor lesions.

A fresh tumor sample will be obtained by biopsy at baseline and theresected specimen will be collected at surgery. Pharmacology (PK/PD) andbiomarker studies will be conducted before and after surgery.

The patients will be followed up to one year after the first cycle ofadministration. After the end of study visit, relapse and survival willbe documented post study according to local practices during 2additional years.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Where“about” is used in connection with a number, this can be specified asincluding values corresponding to +/−10% of the specified number.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

We claim:
 1. A method of treating an individual having a cancer with ananti-NKG2A antibody that binds and neutralizes the inhibitory activityof NKG2A, wherein antibody competes with HLA-E for binding to NKG2A, thetreatment comprising administering to the individual the antibody for atleast one administration cycle in which the anti-NKG2A antibody isadministered at least twice and in amounts effective to maintain betweentwo successive administrations of the anti-NKG2A antibody, a bloodconcentration of anti-NKG2A antibody of at least 10 μg/ml.
 2. The methodof claim 1, wherein the anti-NKG2A antibody is administered at leasttwice and in amounts effective to maintain a continuous bloodconcentration of the anti-NKG2A antibody of at least 10 μg/ml throughoutthe administration cycle.
 3. The method of claim 1, wherein the antibodyis administered to maintain a continuous blood concentration ofanti-NKG2A antibody of at least 10 μg/ml throughout the administrationcycle.
 4. The method of claim 1, wherein the antibody is administered toachieve a peak blood concentration of anti-NKG2A antibody of at least100 μg/ml upon administration.
 5. The method of claim 1, wherein theantibody is administered in an amount effective to provide a continuous(minimal) blood concentration of the anti-NKG2A antibody of at leastabout 100 μg/ml for at least one week following administration of theanti-NKG2A antibody.
 6. The method of claim 1, wherein the antibody isadministered to maintain a continuous (minimal) blood concentration ofthe anti-NKG2A antibody of at least about 100 μg/ml between twosuccessive administrations of the anti-NKG2A antibody.
 7. The method ofclaim 1, wherein the antibody is administered to maintain a continuousblood concentration of the anti-NKG2A antibody of at least 100 μg/mlbetween two successive administrations of the anti-NKG2A antibody,optionally throughout the administration cycle.
 8. The method of claim1, wherein the antibody is administered 2 times per month.
 9. The methodof claim 1, wherein the antibody is administered intravenously two timesper month and the amount of anti-NKG2A antibody administered is between2-10 mg/kg body weight.
 10. The method of claim 1, wherein the antibodyis administered intravenously two times per month and the amount ofanti-NKG2A antibody administered is between 4-10 mg/kg body weight. 11.The method of claim 10, wherein the treatment comprises a loading periodin which the antibody is administered at least once at an initial doseeffective to maintain a blood concentration of at least 100 μg/ml untilthe next successive administration of the anti-NKG2A antibody, followedby a maintenance period in which the antibody is administered at leasttwice in a second dose and at a frequency effective to maintain acontinuous blood concentration of the anti-NKG2A antibody of at least100 μg/ml between successive administrations of the anti-NKG2A antibody.12. The method of claim 11, wherein the antibody is administeredintravenously, and wherein the loading period comprises administeringthe antibody once at a dose of between 8-10 mg/kg, and the maintenanceperiod comprises administering the antibody at least twice, at aninterval of about two weeks at dose of between 2-6 mg/kg body weight.13. The method of claim 1, wherein the individual has a hematologicaltumor.
 14. The method of claim 1, wherein the individual has a solidtumor.
 15. The method of claim 14, wherein the individual has a head andneck squamous cell carcinoma (HNSCC).
 16. The method of claim 1, whereinantibody has an EC50 for binding to NKG2A-expressing cells of between1-10 ng/ml.
 17. The method of claim 1, wherein antibody has KD ofbetween 10-10 M and 10-12 M for binding to a recombinant soluble NKG2Apolypeptide, as assessed by surface plasmon resonance.
 18. The method ofclaim 1, wherein the antibody comprises a human IgG4 constant region,wherein the antibody comprises an Fc-engineered constant regioncomprising an amino acid modification that reduces binding to a humanFcγ receptor, or wherein the antibody fragment lacks an Fc domain. 19.The method of claim 1, wherein the antibody comprises a heavy chaincomprising an amino acid sequence of any one of SEQ ID NO: 2-6 and alight chain comprising the amino acid sequence of SEQ ID NO:
 7. 20. Amethod for the treatment or prevention of a cancer in an individual withan antibody that binds and neutralizes the inhibitory activity of NKG2A,comprising: a) determining the HLA-E polypeptide status of malignantcells within the individual having a cancer, and b) upon a determinationthat the patient has HLA-E polypeptides prominently expressed on thesurface of malignant cells, administering to the individual the in anamount effective to achieve a blood (serum) concentration of theanti-NKG2A antibody that corresponds to at least the EC50, optionallythe EC100, for NKG2A+ NK cell response.